RU2174845C2 - Compositions and methods of genetic material delivery - Google Patents

Abstract

FIELD: medicine, medicinal genetics. SUBSTANCE: method involves introduction of genetic material with enhancer of polynucleotide function (for example, bupivacaine) in cells of individual person. Molecules of nucleic acid no containing retrovirus particles are incorporated also. Nucleic acid molecule has the sequence that encodes protein which involves at least one epitope identical or similar with antigen epitope being the latter is a target for immune response. This nucleotide sequence is bound operatively with regulatory sequences. Invention describes also a pharmaceutical composition and set for method realization. EFFECT: broadened arsenal of agents against HIV infection. 48 cl, 1 tbl, 19 dwg, 59 ex

Description

 The invention relates to preparations and methods for introducing genetic material into cells of an individual. The preparations and methods of the invention can be used to deliver protective and / or therapeutic agents, including genetic material encoding protein targets for vaccination and therapeutic proteins.

 As a means of replacing defective genetic information, the direct introduction of a normally functioning gene into a living organism was considered. In separate studies, DNA is injected directly into the cells of a living organism without the use of a viral particle or other infectious vector. In the work of Nabel E.G. et al. (1990) Science 249, 1285-1288 disclosed a site-specific gene expression in vivo of a beta-galactosidase gene transferred directly to the wall of a mouse artery. In the work of Wolfe J.A. et al. (1990) Science 247, 1465-1468, the expression of a variety of reporter genes transferred directly to mouse muscles in vivo is disclosed. Acsadi G. et al. (1991) Nature 352, 815-818, disclosed expression of a human dystrophin gene in a mouse after intramuscular injection of a DNA construct. Wolfe J. A. et al. (1991) Biotechniques 11 (4), 474-485, which is incorporated herein by reference, refers to conditions affecting direct gene transfer to rodent muscles in vivo. In the work of Felgner P.L. and G. Rhodes (1991) Nature 349, 351-352, disclosed are direct drug delivery of isolated genes without the use of retroviruses.

 The use of direct gene transfer has been proposed as an alternative to the anti-pathogenic vaccination method. As a possible HIV vaccination strategy, the use of direct gene transfer through a single injection is proposed. There are reports of an observed cellular immune response to HIV-gp120 that occurs when plasmid DNA encoding said glycoprotein is introduced into the cell. PCT International Application No. PCT / US 90/01515, published October 4, 1990, discloses methods for vaccinating an individual against a pathogenic infection by directly injecting polynucleotides into individual cells using a one-step technique. The use of transfection agents other than lipofectins is specifically excluded from the disclosed methods. Stimulation of inoculated cells is not disclosed and is not expected. Disclosed is an HIV vaccine consisting of introduced polynucleotides encoding the gp120 viral protein. Evidence of the effectiveness of the vaccine is not given.

 Thomason DB et al. (1990) Cell Physiol, 27, 578-581 and PCT patent application N WO 91/12329 disclose the introduction of bupivacaine (bupivacaina) into muscle cells in order to induce the proliferation of satellite cells as part of the gene delivery technique when participation of a retrovirus.

 The present invention relates to methods for introducing genetic material into cells of an individual. The methods include the steps of contacting the cells of an individual with an agent enhancing the function of the polynucleotide, which is recommended as an agent that facilitates the uptake of DNA by the cell or enhances the inflammatory response, and introducing into the cells nucleic acid molecules containing a nucleotide sequence that either encodes the target peptide or protein, or serves as a matrix for functional nucleic acid molecules. The introduced nucleic acid molecules do not contain retroviral particles. The target protein can be either a protein that functions in the body of an individual, or a protein that serves as a target for an immune response.

 The present invention relates to a method for vaccinating an individual against a pathogen. The method includes the steps of contacting the cells of an individual with an agent enhancing the function of the polynucleotide, which is recommended as an agent that facilitates the absorption of DNA by the cell or enhances the immune response, and introducing into the cells a nucleic acid molecule containing a nucleotide sequence encoding a peptide having at least one epitope that identical or substantially similar to an epitope present on a pathogen antigen and operably linked to regulatory sequences. A nucleic acid molecule is able to be expressed in an individual's cells.

 The present invention relates to a method for vaccinating a person against HIV. The method consists in introducing to a person a nucleic acid molecule containing a nucleotide sequence encoding at least one peptide, which includes at least one epitope that is identical or essentially similar to the epitope present on an HIV protein operably linked to regulatory sequences.

 The present invention relates to a method for vaccinating a person against HIV. The method consists in introducing molecules of two different nucleic acids into different human cells. The molecule of each nucleic acid contains a nucleotide sequence encoding at least one peptide, which includes at least one epitope that is identical or substantially similar to the epitope present on an HIV protein operably linked to regulatory sequences. Each of the molecules of different nucleic acids contains a different nucleotide sequence encoding at least one peptide different from the other, and each nucleic acid is able to be expressed in human cells.

 The present invention relates to a method for vaccinating an individual against hyperproliferative disease or autoimmune diseases. The method consists in introducing into the cells of an individual nucleic acid molecules containing a nucleotide sequence encoding a peptide comprising at least one epitope that is identical or substantially similar to an epitope located in a protein associated with a hyperproliferative disease or a protein associated with an autoimmune a disease, respectively, and operably linked to regulatory sequences, the nucleic acid molecule being able to be expressed in cells.

 The present invention relates to methods for treating an individual suffering from a disease, comprising the steps of contacting the individual's cells with an agent enhancing the function of the polynucleotide, which is recommended as an agent that facilitates the uptake of DNA by cells or enhances the inflammatory response, and introducing into the individual's cells nucleic acid molecules containing a nucleotide sequence that functions instead of a defective gene or a coding molecule that creates a therapeutic effect in the body of an individual and opera closely associated with regulatory sequences, moreover, nucleic acid molecules are able to be expressed in cells.

 The present invention relates to pharmaceutical preparations comprising a nucleic acid and a polynucleotide function enhancer. The present invention relates to a pharmaceutical kit comprising a container with a nucleic acid and a container with a polynucleotide function enhancer.

 The present invention relates to HIV prophylactic and therapeutic vaccines comprising a pharmaceutically acceptable carrier or diluent and a nucleic acid encoding one or more peptides, each of which has at least one epitope that is identical or essentially similar to the epitope found in at least one HIV a protein operably linked to regulatory sequences, the nucleic acid being able to be expressed in human cells.

 The present invention relates to prophylactic and therapeutic HIV vaccines containing two inoculum. The first inoculum consists of a pharmaceutically acceptable carrier or diluent and a first nucleic acid. The first nucleic acid molecule includes a nucleotide sequence encoding one or more peptides, each of which contains at least one epitope identical or substantially similar to the epitope present in at least one HIV protein operably linked to regulatory sequences, the nucleic acid molecule capable of being expressed in human cells. The second inoculum consists of a pharmaceutically acceptable carrier or diluent and a second nucleic acid. The second nucleic acid molecule comprises a nucleotide sequence encoding one or more peptides, each of which contains at least one epitope that is identical or substantially similar to the epitope present in at least one HIV protein operably linked to regulatory sequences, the nucleic acid molecule capable of being expressed in human cells. The molecules of the first and second nucleic acids are different and encode different peptides.

 In FIG. 1A is a diagram illustrating the construction of the plasmid pM160 created by inserting a fragment encoded in PCR (polymerase chain reaction) encoding HIV-HXB2 glycoprotein gp160 into plasmid pMAMneoBlue (Clontech).

 In FIG. Figure 1B shows a photograph of an autoradiogram of Western blotting of the complete cell lysate of cells transfected with plasmid pM160 (3G7 cells), compared with cells transfected with vector only (TE671 cells). One can see the production of gp120 and gp41 in 3G7 cells, but not in TE 671 cells.

In FIG. 2 shows a photograph of an autoradiogram of immunoprecipitation results when serum antibodies bind to ¹²⁵ J-gp160.

 In FIG. 3A-3E are graphs showing ELISA (FISA) results on binding of various sera to various proteins immobilized on microtiter plates.

In FIG. 4A and 4B are photographs of MT-2 cells infected with TCID ₅₀ HIV-1 / III ₈ cell-free virus previously incubated in successive dilutions of antiserum.

In FIG. Figure 4C shows graphs showing the change in neutralization values (V _n / V _o ) depending on dilution factors and based on the results using control serum (x = mice immunized with pMAMneoBlue vector) and test serum (o = mice immunized with pM160).

In FIG. 4D-4G shows photographs of H9 / III of ₈ cells used in experiments to study syncytial inhibition using serum from immunized and control animals.

 In FIG. Figure 5 is a graph depicting the survival of immunized and non-immunized mice that were challenged with gp160-labeled and unlabeled tumor cells. Mice are immunized with the recombinant gp160 protein of only vector DNA or a recombinant vector comprising DNA encoding gp160. Mice are injected with SP2 / 0 tumor cells or SP2 / 0-gp160 tumor cells (SP2 / 0 cells transfected with DNA encoding gp160 and expressing gp160).

 In FIG. 6 shows a map of plasmid pGAGPOL.rev.

 In FIG. 7 shows a map of plasmid pENV.

 In FIG. Figure 8 shows four macromolecular skeletons (A, B, C, and D) used to construct the genetic construct.

 In FIG. 9 shows four inserts (1, 2, 3, and 4) inserted into skeletons to produce genetic constructs.

 The present invention relates to a method for introducing nucleic acid molecules into animal cells, thereby providing a high level of absorption and functioning of nucleic acid molecules. The method of the present invention includes the steps of introducing into the cells of an individual nucleic acid molecules that do not contain viral particles, in particular retroviral particles, in combination with the introduction of a co-agent that enhances the inflammatory response and / or enhances the expression of nucleic acid molecules in the tissues and / or facilitates absorption nucleic acid molecule cells. Recommended embodiments of the present invention provide methods for delivering nucleic acid molecules to individual cells without the use of infectious agents.

 The nucleic acid molecules delivered according to the invention into cells can serve as: 1) genetic matrices for proteins that act as prophylactic and / or therapeutic immunizing agents; 2) copies that replace defective, missing or non-functioning genes; 3) genetic matrices for therapeutic proteins; 4) genetic matrices for antisense molecules; or 5) genetic matrices for ribozymes. In the event that the nucleic acid molecule encodes a protein, it is recommended that the nucleic acid molecule include the sequences necessary for transcription and translation in animal cells. In the case of nucleic acid molecules serving as a matrix for antisense molecules and ribozymes, it is recommended that such nucleic acids be linked to the regulatory elements necessary for the production of a sufficient number of copies of the antisense molecules and ribozymes encoded by them, respectively. The nucleic acid molecules do not contain retroviral particles and are preferably given as plasmid-shaped DNA.

 The co-agent is also called a “polynucleotide function enhancer” or “UVP”. UVP is a compound or mixture of compounds that enhance the inflammatory response and / or enhance the expression of nucleic acid molecules in tissues and / or facilitate the uptake of nucleic acid molecules by cells, preferably having several of these properties. Polynucleotide function enhancers that facilitate the uptake of DNA and RNA by cells and stimulate cell division and replication are also called cell-stimulating agents. The coagents recommended for the present invention are selected from the group consisting of benzoic acid esters and anilides. In recommended embodiments, the UVP is represented by bupivacaine.

 In accordance with some aspects of the present invention, there are provided preparations and methods by which an individual is prophylactically and / or therapeutically vaccinated against a pathogen or abnormal disease-related cell. The genetic material encodes a peptide or protein that shares at least one epitope with an immunogenic protein found in the target pathogen or cells. Genetic material is expressed by the cells of the individual, and serves as an immunogenic target against which an immune response occurs. The resulting immune response has a broad basis: in addition to the humoral immune response, both types of cellular immune responses are also triggered. The methods of the present invention are useful for imparting prophylactic or therapeutic immunity. Thus, the method includes both methods of protecting an individual from infection by a pathogen or the emergence or proliferation of specific cells, and methods of treating an individual suffering from infection by a pathogen, hyperproliferative disease or autoimmune disease.

 The present invention allows to induce a wide range of immune responses against target proteins, i.e. proteins specifically associated with pathogens or the individual's own "abnormal" cells. The present invention is applicable for vaccinating individuals against pathogenic agents or microorganisms in such a way that the immune response against the pathogen provides protective immunity against the pathogen. The present invention is useful for controlling hyperproliferative diseases and disorders, for example, cancer, by creating an immune response against a target protein specifically associated with hyperproliferative cells. The present invention allows the fight against autoimmune diseases and disorders by creating an immune response against the target protein specifically associated with cells responsible for the autoimmune condition.

 Some aspects of the present invention relate to gene therapy, i.e., nucleic acid preparations and methods for their introduction into the cells of an individual in the form of exogenous ion ions of a gene that either corresponds to a defective, missing, non-functioning or partially functioning gene of the individual, or encodes therapeutic proteins, t .e. proteins whose presence in the body of the individual compensates for their deficiency in the body and / or the presence of which creates a therapeutic effect on the body of the individual. Thus, the present invention provides protein delivery vehicles in an alternative way than simple administration of the protein.

 It is understood that, as used herein, the term “target protein” refers to peptides and proteins encoded by the gene constructs of the present invention, which either serve as protein targets for the immune response, or act as therapeutic or compensating proteins in gene therapy regimens.

 According to the present invention, DNA and RNA encoding a target protein are introduced into the cells of an individual, where it is expressed and produced as a result of the target protein. DNA or RNA encoding a target protein is bound to regulatory elements necessary for expression in an individual's cells. Regulatory elements for DNA expression include a promoter and a polyadenylation signal. Other elements, such as the Kozak region, may also be included in the genetic construct.

 As used herein, the term "genetic construct" refers to a DNA or RNA molecule containing a nucleotide sequence encoding a target protein, and which includes initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression into cells of the vaccinated individual.

 As used herein, the term “expressed form” refers to a gene construct containing the necessary regulatory elements operably linked to a coding sequence such that when they are present in an individual’s cells, the coding sequence is expressed. The coding sequence encodes the target protein.

 As used herein, the term “genetic vaccine” refers to a pharmaceutical preparation containing a genetic construct. Such a construct includes a nucleotide sequence encoding a target protein. These drugs include pharmaceuticals that are useful for creating a therapeutic immune response.

 As used herein, the term “genetic therapeutic (therapeutic) drug” refers to a pharmaceutical preparation containing a genetic construct. The construct includes a nucleotide sequence encoding a therapeutic or compensating protein.

 As used herein, the term “target protein” refers to a protein against which an immune response can be caused. The target protein is an immunogenic protein sharing at least one epitope with a protein from a pathogen or cell of an undesired type, for example a cancer cell or a cell participating in an autoimmune disease against which vaccination is required. An immune response directed against the target protein can protect the individual from the specific infections or diseases with which the target protein is associated, or can cure them.

 As used herein, the term “separate epitope” refers to proteins containing at least one epitope that is identical or substantially similar to an epitope of another protein.

 It is understood that, as used herein, the expression "substantially similar to an epitope" refers to an epitope whose structure is not identical to that of a protein epitope, but nonetheless capable of inducing a cross-cell or humoral immune response.

 As used herein, the term “therapeutic (therapeutic) protein” refers to proteins whose presence has a therapeutic effect on an individual.

 It is believed that, as used herein, the term “compensating protein” will refer to proteins whose presence compensates for the absence of a fully functional, endogenously produced protein associated with the absence, defect, nonfunctionality or partial functionality of the endogenous gene.

 Genetic constructs include a nucleotide sequence encoding a target protein operably linked to regulatory elements necessary for gene expression. Accordingly, the introduction of a DNA or RNA molecule into a living cell results in the expression of DNA or RNA encoding the target protein, thereby producing the target protein.

 When a cell assimilates a genetic construct, including a nucleotide sequence encoding a target protein operably linked to regulatory elements, it can remain in the cell as a functioning extrachromosomal molecule or can be integrated into the chromosomal DNA of the cell. DNA can be introduced into the cell, where it will remain as a separate genetic material in the form of a plasmid. Or linear DNA that can integrate with the chromosome can be introduced into the cell. When DNA is introduced into the cell, reagents can be added to facilitate the integration of DNA with the chromosomes. DNA sequences that can promote integration can also be included in the DNA molecule. Alternatively, RNA can be introduced into the cell. It is also possible the creation of a genetic construct in the form of a linear minichromosome, including centromeres, telomeres, and the origin of replication.

 The molecule encoding the target protein may be DNA or RNA containing the nucleotide sequence encoding the target protein. Such molecules can be represented by cDNA, genomic DNA, synthetic DNA or a hybrid thereof, or an RNA molecule, for example mRNA. Accordingly, it is believed that, as used herein, the terms “DNA construct”, “genetic construct” and “nucleotide sequence” refer to both DNA and RNA molecules.

Regulatory elements necessary for gene expression of a DNA molecule include: a promoter, an initiation codon, a termination codon, and a polyadenylation signal. In addition, amplifiers are often required for gene expression. It is necessary that such elements are operably linked to the sequence encoding the target proteins and that regulatory elements are operable in the body of the individual to whom they are introduced
It is generally believed that the initiation codon and the termination codon are part of the nucleotide sequence encoding the target protein. However, it is necessary that these elements are functional in the body of the individual to whom the gene construct is introduced. The initiating and terminating codons must be in the same frame with the coding sequence.

 The promoters and polyadenylation signals that are used must be functional within the cell of an individual.

 Examples of promoters useful in the practice of the present invention, especially in the preparation of a human genetic vaccine, include, but are not limited to, promoters from monkey virus 40 (OB40), mouse breast tumor virus (VOMG) promoter, human immunodeficiency virus (HIV) for example, HIV long terminal repeat (DCT) promoter, Moloni virus, ALV, cytomegalovirus (CMV), e.g. CMV directly early promoter, Epstein-Barr virus (EBV), Routh sarcoma virus (HRV), as well as promoters from human genes, for example actin and human, human myosin, human hemoglobin, human muscle creatine, and metallothionein.

 Examples of polyadenylation signals useful in the practice of the present invention, especially when producing a human vaccine, include (but are not limited to) OB40 polyadenylation signals and DCT polyadenylation signals. In particular, an OB40 polyadenylation signal is used, which is a pCEP4 plasmid (Invitrogen, San Diego, CA), called an OB40 polyadenylation signal.

 In addition to the regulatory elements necessary for the expression of DNA, other elements may also be included in the DNA molecule. Also additional elements include amplifiers. The amplifier may be selected from the group including (but not limited to) human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers, for example, amplifiers from CMV, HRV and EBV.

 Genetic constructs can be provided by the origin of mammalian replication, the purpose of which is to keep the construct outside the chromosome and obtain multiple copies of the construct in the cell. Invitrogen (San Diego, CA) plasmids pCEP4 and pREP4 contain the Epstein-Barr virus origin of replication and the coding region of the nuclear antigen EBNA-1, which provides high-copy episomal replication without integration.

 In some recommended embodiments of the invention, the vector used is selected from the vectors described in Example 46. In those aspects of the invention that relate to gene therapy, constructs with an origin of replication including the antigen necessary for activation are recommended.

 In some recommended vaccination-related embodiments of the invention, the genetic construct contains nucleotide sequences encoding a target protein, and furthermore includes genes for proteins that enhance the immune response against such target proteins. Examples of such genes include genes encoding cytokines and lymphokines, for example alpha interferon, gamma interferon, platelet derived growth factor (PTFR), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12. In some embodiments, it is recommended that the gene for GM-CSF be included in the genetic constructs used in vaccine preparations.

 An additional element can be added that serves as a target for cell destruction, if for any reason it is desirable to exclude the entry of a genetic construct into the cell. A herpes thymidine kinase (tk) gene may be included in the genetic construct. The drug gangciclovir may be administered to an individual, leading to the selective destruction of any tk-producing cells, thereby providing means for the selective destruction of cells with a genetic construct.

 To maximize protein production, regulatory sequences that are well matched to gene expression in cells into which the designer was introduced can be selected. In addition, codons that are most efficiently transcribed in cells can be selected. Any specialist is able to create constructs that function in DNA cells.

 To detect expression, genetic constructs can be analyzed in vltro for the expression level using tissue cultures of cells of the same type as the cells into which the construct will be introduced. For example, if a genetic vaccine is to be introduced into human muscle cells, muscle cells grown in culture, for example, solid muscle tumor cells of rhabdomyosarcoma, can be used to determine the expression level as an in vitro model.

 The genetic constructs used in the present invention are not introduced into retroviral particles. Genetic constructs are absorbed by the cell without the participation of a retroviral particle insert, such as that which occurs when retroviral particles with RNA introduced into them infect the cell. As used herein, the term “non-retroviral particles” refers to genetic constructs that are not inserted into retroviral particles. As used herein, the expression "separated from an infectious agent" refers to genetic material that is not part of a viral, bacterial or eukaryotic vector (active, inactivated, alive or dead) capable of infecting a cell.

 In some embodiments of the invention, the genetic constructs are not sufficiently complete replicable viral genomes, as a result of which, when introduced into the cell, the genetic construct has enough genetic information to ensure the production of infectious viral particles. As used herein, the term "incomplete viral genome" refers to a genetic construct containing an insufficiently complete genome, and therefore, when such a genetic construct is introduced into the cell, no genetic information is sufficient to produce an infectious virus.

 In some embodiments, the attenuated viral vaccine may be delivered as a genetic construct containing enough genetic material to allow the production of viral particles. The delivery of an attenuated vaccine in the form of a genetic construct appears to be an easier way to produce large quantities of a safe, pure active vaccinating product.

 The genetic construct can be introduced with or without the use of "microprojectiles". An advantage is that the genetic construct of the present invention can be delivered to individual cells without particulate matter. It is understood that the expression “without particulate matter” as used herein refers to a fluid that does not contain any solid “microprojectiles” used as a means for perforating, puncturing, or otherwise piercing the cell membrane to create an inlet for the entry of genetic material into the cell.

 The present invention can be used to vaccinate an individual against all pathogens, for example viruses, prokaryotic and pathogenic eukaryotic microorganisms, such as unicellular pathogenic microorganisms and multicellular parasites. The present invention is particularly suitable for vaccinating an individual against pathogens that infect a cell and that are not encapsulated, such as viruses and prokaryotes, such as gonorrhea, listeriosis and shigellosis. In addition, the present invention is also applicable for vaccinating an individual against pathogens from the class of protozoa, in the life cycle of which there is a stage when they are intracellular pathogens. It is understood that, as used herein, the term "intracellular pathogen" refers to a virus and a pathogenic microorganism that spend at least part of its reproductive or life cycle in a host cell in which pathogenic proteins are produced or forced to produce. Table 1 lists some of the viral families and genera for which the vaccines of the present invention can be made. In DNA vaccines, DNA constructs can be used that include DNA sequences encoding peptides that contain at least one epitope that is identical or substantially similar to the epitope present on a pathogen antigen, for example, those antigens listed in the tables. Moreover, the present invention is also applicable for vaccinating an individual against other pathogens, including prokaryotic and eukaryotic pathogens from the class of protozoa, as well as multicellular parasites, for example those listed in Table 2.

 To create a genetic vaccine designed to protect against pathogenic infection, genetic material encoding immunogenic proteins against which a protective immune response can be created must be included in the genetic construct. Whether the infection is an intracellular pathogen, which is particularly suitable for the present invention, or extracellular, it is unlikely that all pathogen antigens would produce a protective response. Since both DNA and RNA are relatively small and can be obtained relatively easily, the present invention has the additional advantage that it allows vaccination with a number of pathogen antigens. The genetic construct used in a genetic vaccine may include genetic material encoding a variety of antigen pathogens. For example, several viral genes can be included in a single construct, thereby providing multiple targets. In addition, a number of inoculums can be prepared that can be delivered to various cells, and together including in some cases a complete or, more preferably, incomplete, for example almost complete, set of genes in the vaccine. For example, a complete set of viral genes can be introduced using two constructs, each of which contains different half of the genome, introduced in different areas. Thus, an immune response can be created against each antigen without the risk of infectious virus involvement. This allows you to enter more than one antigen target, and also eliminates the requirement to identify protective antigens.

 The ease of handling and the inexpensive nature of DNA and RNA make it possible, moreover, to select protective antigens more effectively. Genes can be sorted and systematically analyzed more easily than proteins. Microorganisms are selected as pathogenic agents to protect against which a vaccine is created, and then an immunogenic protein is identified. Tables 1 and 2 include lists of some pathogenic agents and microorganisms for which genetic vaccines can be created to protect against infection. In some recommended embodiments, methods for vaccinating an individual against a pathogen are directed against HIV, HTLV, or EBV.

 Another aspect of the present invention is a method for imparting a broad-based protective immune response against hyperproliferative cells characteristic of hyperproliferative diseases, and a method for treating an individual suffering from a hyperproliferative disease. It is understood that, as used herein, the term "hyperproliferative disease" refers to those diseases and disorders that are characterized by hyperproliferation of cells. Examples of hyperproliferative diseases include all types of cancer and psoriasis.

 It has been found that the introduction of a genetic construct comprising a nucleotide sequence encoding an immunogenic protein associated with a “hyperproliferating cell” into an individual cell leads to the production of these proteins in the vaccinated cells of the individual. It is understood that, as used herein, the term “hyperproliferatively associated protein” refers to proteins associated with a hyperproliferative disease. For vaccination against hyperproliferative diseases, a genetic construct is introduced to an individual, including a nucleotide sequence encoding a protein associated with a hyperproliferative disease.

 In order for a hyperproliferatively associated protein to be an effective immunogenic target, it must be a protein produced exclusively or at a higher level than in normal cells in hyperproliferative cells. Target antigens include those proteins, fragments thereof, and peptides that contain at least one epitope found in said proteins. In some cases, a hyperproliferatively associated protein is a product of a mutation of a protein-coding gene. A mutated gene encodes a protein that is almost identical to a normal protein, with the exception of some differences in the amino acid sequence, which leads to an excellent epitope not found in a normal protein. Such target proteins include those encoded by oncogenes, for example: myb, myc, fyn, as well as the translational gene bcr / ab1, ras, src, P53, neu, trk and ECRF. In addition to oncogenic products, target proteins for anticancer treatment and protective regimes include variable regions of antibodies produced by B-cell lymphomas and variable regions of T-cell receptors from T-cell lymphomas, and in some embodiments, targeted antigens for autoimmune diseases are also used . Other proteins associated with the tumor can be used as target proteins, for example, proteins found at the highest level in tumor cells, including proteins recognized by monoclonal antibody 17-1A and folate binding proteins.

 Although the present invention can be used to vaccinate an individual against one or more forms of cancer, nevertheless, the present invention is particularly suitable for the prophylactic vaccination of an individual predisposed to the development of a particular type of cancer or having cancer, thereby being prone to relapse. Advances in genetics and technology, as well as epidemiology, help determine the likelihood and risk of developing cancer in an individual. By using genetic selection and / or family histories, one can predict the likelihood of a particular individual developing one of several types of cancer.

 Similarly, those individuals who have already had cancer and who have been treated with the removal of the tumor, or for some other reason in remission, are especially prone to relapse and re-manifestations of the cancer. As part of the treatment process, such individuals can be vaccinated against the cancer that was diagnosed with them, in order to combat its repeated manifestations. Thus, after it has become known that an individual has a cancer of some type and there is a risk of relapse, this individual can be vaccinated in order to prepare his immune system to fight any future manifestations of cancer.

 The present invention provides a method of treating an individual suffering from a hyperproliferative disease. In such methods, the introduction of genetic constructs serves immunotherapeutic, directing and immune system promoting purposes in order to combat producing hyperproliferative cells.

 The present invention provides a method of treating individuals suffering from autoimmune diseases and disorders by imparting a broad-based immune response against targets associated with autoimmunity, including cell receptors and self-directed antibodies producing cells.

 Autoimmune diseases transmitted through T cells include: rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, polio dermatitis, scleroderma, dermatitis and ulcerative colitis. Each of these diseases is characterized by the participation of T-cell receptors that bind to endogenous antigens and initiate the inflammatory cascade associated with autoimmune diseases. Vaccination against a variable region of T cells can cause an immune response, including CTL, to remove these T cells.

In the case of RA, several specific variable regions of T-cell receptors (TCRs) involved in the disease have been characterized. Similar TCRs include: V _β -3, V _β -14, V _β -17 and V _α -17. Thus, vaccination of DNA with a construct encoding at least one of these proteins will elicit an immune response directed at the T cells involved in RA. See: Howell MD et al., 1991, Proc. Natl. Acad Sci. US 88, 10,821-10925; Palliard X. et al., 1991, Science 253, 325-329; Williams WV et al., 1992, J.Cin.Invest. 90, 326-333; each of these works is introduced here as a reference.

In the case of MS, several specific variable regions of TCR involved in the disease have been characterized. Such TCRs include: V _β -7 and V _α -10. Thus, DNA vaccination with a construct encoding at least one of these proteins creates an immune response directed at T cells participating in MS. See: Wucherphennig KW et al., 1990, Science 248, 1016-1019; Oksenberg JR et al., 1990, Nature 345, 344-346; each of these works is introduced as a reference.

Таким образом, вакцинации ДНК конструктом, кодирующим по меньшей мере один из указанных белков, создаст иммунную реакцию, направленную на участвующие в склеродермии Т-клетки.In the case of scleroderma, several specific variable regions of TCR involved in the disease have been characterized. These TCRs include:

Thus, vaccination of DNA with a construct encoding at least one of these proteins will create an immune response directed at T cells involved in scleroderma.

 In the treatment of patients suffering from an autoimmune disease transmitted through T cells, in particular patients for whom variable regions of TCR have not yet been characterized, a synovial biopsy can be performed. Samples of T cells present can be taken in which, using standard techniques, the variable regions of these TCRs are identified. Based on the information received, genetic vaccines can be prepared.

 Autoimmune diseases transmitted through B cells include lupus (SLE), severe myasthenia gravis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosis, and pernicious anemia. Each of these diseases is characterized by antibodies that bind to endogenous antigens and initiate an inflammatory cascade associated with autoimmune diseases. Vaccination against variable regions of antibodies can induce an immune response, including CTL, with the removal of antibody-producing B cells.

 To treat a patient suffering from B-cell transmitted autoimmune disease, it is necessary to identify a variable region of antibodies exhibiting autoimmune activity. A biopsy can be performed with sampling of antibodies present at the site of inflammation. The variable region of these antibodies can be identified using standard techniques. Based on the information received, genetic vaccines can be prepared.

 In the case of SLE, one of the antigens is believed to be DNA. Thus, in patients subject to vaccination against SLE, their serum can be analyzed for anti-DNA antibodies and a vaccine can be prepared that includes DNA constructs encoding a variable region of those found in the serum of anti-DNA antibodies.

 Common structural features for variable regions of both TCR and antibodies are well known. A DNA sequence encoding specific TKRs and an antibody can typically be detected using well-known methods described, for example, in Kabat et al., 1987, "Sequences of Immunological Interest of Proteins", USA, Department of Human Health and Human Services , Bethesda, MD, which is incorporated herein by reference. In addition, a general method for cloning functional variable regions from antibodies can be found in: Chaudhery V.K. et al., 1990, Proc. Natl.Acad. Sci. US 87, 1066, which is incorporated herein by reference.

 In some embodiments of the invention related to gene therapy, gene constructs contain either compensating genes or genes encoding therapeutic proteins. Examples of compensating genes include a gene encoding dystrophin or a functional fragment thereof, a gene intended to compensate for a defective gene in patients with cystic fibrosis, insulin, a gene for compensating for a defective gene in patients suffering from ADA, and a gene encoding factor VIII. Examples of genes encoding therapeutic proteins include genes encoding erythropoietin, interferon, LDL receptor, GM-CSF, IL-2, IL-4 and TNF. In addition, genetic constructs encoding single chain antibody components that specifically bind to toxic substances can be introduced.

In some recommended embodiments, the dystrophin gene is given as a minigene, which is used to treat individuals suffering from muscular dystrophy. In certain recommended embodiments, a minigen is provided containing a sequence encoding a portion of a dystrophin protein. Disturbances in the dystrophin gene lead to Becker softer muscular dystrophy
(MDB), and to more severe muscular dystrophy Duchenne (DMD) In the case of MDB, dystrophin is formed, but has deviations from the norm in size and / or in quantity. The patient is in a state of mild to moderate. With DMD, protein is not formed, and the patient is confined to a chair at the age of 13 and usually dies at the age of 20. In some patients, in particular MDB patients, a partial dystrophin protein obtained by expression of the minigen provided by the method of the invention can lead to an improvement in muscle function.

 In some recommended embodiments, genes encoding IL-2, IL-4, interferon, or TNF are delivered to tumor cells that are either present or removed and then returned to the individual. In certain embodiments, a gene coding for gamma interferon is administered to an individual suffering from multiple sclerosis.

 Antisense molecules and ribozymes can also be delivered to individual cells by introducing genetic material that acts as a template for copies of such active agents. Such agents deactivate or otherwise affect the expression of genes encoding proteins whose presence is undesirable. Constructs containing sequences encoding antisense molecules can be used to inhibit or prevent the production of proteins in a cell. Thus, the production of proteins such as oncogen products can be eliminated or reduced. Similarly, ribozymes can interrupt gene expression by selective destruction of messenger RNA before it is translated into protein. In some embodiments, cells are treated by the method of the invention using constructs encoding antisense molecules or ribozymes as part of a treatment process, including administration of other drugs and other procedures. In gene constructs encoding antisense molecules and ribozymes, vectors similar to those used when protein production is desired are used, except that the coding sequence does not have an initiation codon that initiates RNA translation into protein. In some embodiments, it is recommended that the vectors described, in particular in Example 46, contain the origin of replication and the expressed form of the corresponding nuclear antigen.

 Ribozymes are catalytic RNAs capable of self-cleaving or cleaving molecules of other RNAs. Ribozymes of several different types are known to those skilled in the art, for example, the hammer type, hairpin, group I intron Tetrahymena, ax and RNase P (S. Edgington, Biotechnology, 1992, 10, 256-262). Hammer-type ribozymes possess a catalytic site that was mapped to the nucleus in less than 40 nucleotides. Some ribozymes in plant viroids and satellite RNAs share a common secondary structure and certain conserved nucleotides. Although these ribozymes in nature act as their own substrate, the substrate domain can be directed to another RNA substrate by base pairing in sequences flanking the preserved cleavage site. This ability of ribozymes of the usual structure allows their use for sequence-specific cleavage of RNA (G. Peolella et al., EMBO 1992, 1913-1919). Thus, it is within the competence of a specialist to use various catalytic sequences from different types of ribozymes, for example, a catalytic sequence such as a hammer, and construct them in the above manner. Ribozymes can be created that target a variety of purposes, including pathogenic nucleotide sequences and oncogenic sequences. Certain recommended embodiments of the invention include sufficient complementarity to the specifically targeted abl-bcr fusion transcript while maintaining the efficiency of the cleavage reaction.

 In accordance with some embodiments of the present invention, the cells are treated with compounds that facilitate the absorption of the genetic construct by the cell. According to certain embodiments of the invention, the cells are treated with compounds that facilitate cell division and promote uptake of the genetic construct. The introduction of compounds that facilitate the uptake of the genetic construct by the cell, including those stimulating the cell, leads to a more effective immune response against the target protein encoded by the genetic construct.

 According to some embodiments of the present invention, the genetic construct is introduced into the body of an individual using a needleless injection device. In accordance with some embodiments of the present invention, the genetic construct is introduced simultaneously into the body of an individual intradermally, subcutaneously and intramuscularly using a needleless injection device. Needleless injection devices are well known and widely available. An ordinary specialist, guided by this description, uses a needleless device for injection to supply genetic material to the cells of an individual. Needle-free injection devices are well-suited for delivering genetic material to all tissues. The devices are particularly suitable for delivering genetic material to skin and muscle cells. In some embodiments, a needleless injection device may be used to advance a liquid containing a DNA molecule to the surface of an individual's skin. The liquid advances at a sufficient speed so that when it hits the skin, the liquid penetrates the surface of the skin and passes into the underlying tissues of the skin and muscles. Thus, genetic material is introduced simultaneously percutaneously, subcutaneously and intramuscularly. In some embodiments, a needleless injection device may be used to deliver genetic material to the tissues of another organ in order to introduce a nucleic acid molecule into the cells of that organ.

 According to the invention, the genetic vaccine can be introduced directly into the body of the individual to be vaccinated, or it can be introduced ex vivo into the remote cells of the individual, followed by reimplantation after administration. With any route of administration, genetic material is introduced into cells present in the individual’s body. Routes of administration include, but are not limited to: intramuscular, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular and oral, as well as percutaneous, as an inhalation or suppository. Recommended routes of administration include intramuscular, intraperitoneal, intradermal, and subcutaneous injections. The supply of gene constructs encoding target proteins can confer protective properties to the mucous membranes of an individual if the administration is carried out in a manner in which the material is present in the tissues associated with the protective properties of the mucous membranes. So, in some examples, the gene construct is supplied by insertion into the buccal pocket located in the mouth of the individual.

 Genetic constructs can be introduced using (but not limited to) traditional syringes, needleless injection devices, or "microbombing gene guns." Or, a genetic vaccine can be introduced in a variety of ways into cells removed from the body of an individual. Such methods include, for example, ex vivo transfection, electroporation, microinjection and microbombing. After the cells are absorbed by the gene construct, they are again implanted into the body of the individual. It is believed that usually non-immunogenic cells, after the introduction of gene constructs into them, can be implanted into the individual’s body, even if the vaccinated cells were initially taken from another individual.

 Genetic vaccines of the invention contain DNA in the range from 1 ng to 1000 μg. In some recommended embodiments, the vaccine contains DNA ranging from 10 ng to 800 mcg. In certain recommended embodiments, the vaccine contains 0.1-500 μg of DNA. In other recommended embodiments, the vaccines contain 1-350 μg of DNA. And in yet another recommended embodiment, vaccines contain 25-250 μg of DNA. In other recommended embodiments, the vaccines contain about 100 μg of DNA.

 Genetic vaccines of the present invention are prepared depending on the intended route of administration. An ordinary specialist can easily prepare a genetic vaccine containing a genetic construct. If intramuscular injections are chosen as the route of administration, isotonic preparations are recommended. Used to impart isotonicity additives, as a rule, include: sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are recommended. In some embodiments, a vasoconstrictor agent is added to the preparation. Stabilizers include gelatin and albumin. The pharmaceutical preparations of the present invention are sterile and pyrogen free.

 Genetic constructs of the invention are prepared in the form of a preparation or administered in combination with a polynucleotide function enhancer. Recommended coagents of the present invention are selected from the group consisting of: esters, anilides, amidines, urethanes of benzoic acid and their hydrochlorides, which belong, for example, to the family of local anesthetics.

UVP may be a compound represented by one of the following formulas:
Ar-R ¹ -OR ² -R ³ ,
or
Ar-NR ¹ -R ² -R ³ ,
or
R ⁴ -NR ⁵ -R ⁶ ,
or
R ⁴ -OR ¹ -NR ⁷ ,
where Ar represents benzene, p-aminobenzene, m-aminobenzene, o-aminobenzene, substituted benzene, substituted p-aminobenzene, substituted m-aminobenzene, substituted o-aminobenzene, where the amino group in the aminobenzene derivatives may be represented by amino, C ₁ -C _5- alkylamino, di (C ₁ -C ₅ -alkyl) amino, and substituents in the substituted derivatives are represented by halogen, C ₁ -C ₅ -alkyl and C ₁ -C ₅ -alkoxy;
R ¹ represents C = O;
R ² is C ₁ -C ₁₀ alkyl, including branched alkyls;
R ³ represents hydrogen, amine, C ₁ -C ₅ -alkylamine, di (C ₁ -C ₅ -alkyl) amine,
R ² + R ³ can form cyclic alkyl, C ₁ -C ₅ - alkyl substituted cyclic alkyl, cyclic aliphatic amine, C ₁ -C ₁₀ -alkyl substituted cyclic aliphatic amine, heterocycle, C ₁ -C ₁₀ -alkyl-substituted heterocycle, including C ₁ -C ₁₀ ) -alkyl-N-substituted heterocycle;
R ⁴ represents Ar, R ² or a C ₁ -C ₅ alkoxy group, cyclic alkyl, C ₁ -C ₁₀ -alkyl-substituted cyclic alkyl, cyclic aliphatic amine, C ₁ -C ₁₀ -alkyl-substituted cyclic aliphatic amine, heterocycle, C ₅ -C _{A 10-} alkyl substituted heterocycle and a C ₁ -C ₁₀ alkoxy substituted heterocycle, including a C ₁ -C ₁₀ alkyl-N-substituted heterocycle;
R ⁵ represents C = NH;
R ⁶ is Ar, R ² or a C ₁ -C ₁₀ alkoxy group, cyclic alkyl, C ₁ -C ₁₀ alkyl substituted cyclic alkyl, cyclic aliphatic amine, C ₁ -C ₁₀ alkyl substituted cyclic aliphatic amine, heterocycle, C ₁ -C _{A 10-} alkyl substituted heterocycle and a C ₁ -C ₁₀ alkoxy-substituted heterocycle, including a C ₁ -C ₁₀ -alkyl-N-substituted heterocycle and
R ⁷ represents Ar, R ² or a C ₁ -C ₅ alkoxy group, cyclic alkyl, C ₁ -C ₁₀ alkyl substituted cyclic alkyl, cyclic aliphatic amine, C ₁ -C ₁₀ alkyl substituted cyclic aliphatic amine, heterocycle, C ₁ -C _{A 10-} alkyl substituted heterocycle; and a C ₁ -C ₁₀ alkoxy substituted heterocycle, including a C ₁ -C ₁₀ alkyl-N-substituted heterocycle.

 Examples of esters include benzoic acid esters, for example piperocaine, meprilcaine and isobucaine, para-aminobenzoic acid esters, for example procaine, tetracaine, butetamine, propoxicaine and chlorprocaine; esters of meta-aminobenzoic acid, including metabutamine and primacaine; esters of p-ethoxybenzoic acid, for example parethoxicaine. Examples of anilides include: lidocaine, ethidocaine, mepivacaine, bupivacaine, pyrrocaine and prilocaine. Other examples of such compounds include: dibucaine, benzocaine, diclonin, pramoxin, proparacaine, butacaine, benoxinate, carbocaine, methylbupivacaine, butazine picrate, phenacaine, diotan, luccaine, intracaine, nupercaine, metabutoxicaine, pyridocaine, biphenamine, and biphenamine for example cocaine, cinamoylcocaine, truxillin and cocaethylene and all of these compounds in the form of hydrochlorides.

In recommended embodiments, the UVP is represented by bupivacaine. The difference between bipivacaine and mepivacaine is that bupivacaine has an N-butyl group instead of the N-methyl group of mepivacaine. In this case, we are talking about N-C ₁ -C ₁₀ - alkyl derivatives. The compounds may be substituted with halogen, as is the case with procaine and chlorprocaine. Anilides are more preferred.

 Bupivacaine is administered before, simultaneously with, or after a genetic construct. Bupivacaine and the genetic construct can be prepared as a joint preparation. Bupivacaine is especially useful as a cell-stimulating agent, due to the manifestation of its activity and its diverse properties when introduced into the tissue. Bupivacaine promotes and facilitates the absorption of genetic material by the cell. Bupivacaine per se is a transfection agent. The introduction of genetic constructs together with bupivacaine facilitates the entry of the genetic construct into the cell. It is believed that bupivacaine breaks the cell, or in some other way makes the cell more permeable. Bupivacaine stimulates cell division and replication. Therefore, bupivacaine acts as a replicating agent. The administration of bupivacaine also irritates and damages tissues. As such, bupivacaine acts as an inflammatory agent that causes migration and chemotaxis of immune cells at the injection site. In addition to the cells that are usually present at the injection site, cells of the immune system that migrate as an immune response to the inflammatory agent can come into contact with the introduced genetic material and bupivacaine. Bupivacaine, acting as a transfection agent, is also able to promote the absorption of genetic material by such cells of the immune system.

 Chemically and pharmacologically, bupivacaine is an aminoacyl local anesthetic. Bupivacaine is a homologue of mepivacaine and is related to lidocaine. Bupivacaine gives muscle tissue sensitivity to voltage when sodium is introduced and affects the concentration of ions in the cell. A full description of the pharmacological activity of bupivacaine can be found in the publication: Ritchie J. M. and N.M. Greene, Pharmacological Basics of Therapy, ed. Gilman A.G. et al., 8th edition, chapter 15, p. 3111, which is incorporated herein by reference. In the method of the present invention, the use of bupivacaine and functionally similar compounds of bupivacaine is recommended.

 Bupivacaine-HCl has the following chemical name: 2-piperidinecarboxamide, 1-butyl-N- (2,6-dimethylphenyl) monohydrochloride, monohydrate, and is widely available for pharmaceutical applications by many companies, including Astra Pharmaceutical Products Inc. (Westborough, MA), Sanofi Winthrop Pharmaceuticals (New York, NY), Eastman Kodak (Rochester, NY). Bupivacaine comes on sale with or without methyl paraben, and with or without epinephrine. Any such preparations may be used. For pharmaceutical applications, bupivacaine is marketed at a concentration of 0.25%, 0.5%, and 0.75%, and as such can be used in the invention. Or, if desired, alternative ones that create the necessary concentration effect, in particular 0.05-1%, can be used. According to the present invention, bupivacaine is administered in an amount of 250 μg to 10 mg. In some embodiments, 250 μg to 7.5 mg of bupivacaine is administered. In certain embodiments, 0.05-5 mg of bupivacaine is administered. In other embodiments, bupivacaine is administered in an amount of 0.5-3 mg. And in some embodiments, 5-50 μg of bupivacaine is administered. For example, in some embodiments, 50 μl to 2 ml, preferably 50-1500 μl, more preferably about 1 ml of 0.5% bupivacaine-HCl and 0.1% methylparaben in an isotonic pharmaceutical carrier, are administered simultaneously, or after the introduction of the vaccine. Similarly, in certain embodiments, 50 μl to 2 ml, preferably 50-1500 μl, and more preferably about 1 ml of 0.5% bupivacaine-HCl in an isotonic pharmaceutical carrier, is administered before, simultaneously with or after administration of the vaccine in the same area as the vaccine. Bupivacaine and any other similarly acting compounds, in particular compounds belonging to the family of local anesthetics, can be introduced in concentrations that provide the necessary facilitation of the absorption of genetic constructs by the cell.

 In some embodiments of the invention, the individual is first given an injection of bupivacaine prior to the genetic vaccination by intramuscular injection, i.e. ranging from a week to ten days, for example, an individual is first given an injection of bupivacaine. In some embodiments, an injection of bupivacaine is administered to an individual 1-5 days before the introduction of the genetic construct before vaccination. In certain embodiments, prior to vaccination, approximately 24 hours before the introduction of the genetic construct, the individual is given an injection of bupivacaine. Or bupivacaine can be injected at the same time, several minutes before or after vaccination. Accordingly, bupivacaine and the genetic construct can be combined and injected simultaneously as a mixture. In some embodiments, bupivacaine is administered after the introduction of the genetic construct. For example, an individual is given an injection of bupivacaine from a week to ten days after the introduction of the genetic construct. In some embodiments, the individual is given an injection of bupivacaine approximately 24 hours after vaccination. In certain embodiments, a bupivacaine injection is administered to an individual 1-5 days after vaccination. In other embodiments, bupivacaine is administered to the individual from a week to ten days after vaccination.

 Other agents capable of performing the role of transfecting agents and / or replicating agents and / or inflammatory agents, and which may be administered together with bupivacaine and similarly active compounds, include: pectins, growth factors, cytokines and lymphokines, for example: α-interferon, gamma interferon, platelet derived growth factor (PTFR), GC-SF, GM-CSF, TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10 and IL-12, as well as collagenase, fibroblast growth factor, estrogen, dexamethasone, saponins, surface-active substances such as immunostimulatory complexes (ISCOM), Freund's incomplete adjuvant, LPS analogue, including monophosphoryl lipid A (MPL), muramyl peptides, quinone analogs and vesicles, for example, squalane and svalen, hyaluronic acid and hyaluronidase. All of these substances can be administered in combination with bupivacaine and the genetic construct. For example, bupivacaine and either hyaluronic acid or hyaluronidase are administered together with the genetic construct.

 The genetic construct can be mixed with collagen in the form of an emulsion and introduced parenterally. A collagen emulsion provides a means for the gradual isolation of DNA. Collagen is used in an amount of 50 μl to 2 ml. In a recommended embodiment, using this composition, about 100 μg of DNA is mixed with 2 ml of collagen. Other extended-release drugs may also be used, as described, for example, in the Remington Pharmacist Guide (A. Osol), a standard reference in the art, which is incorporated herein by reference. Such preparations include: aqueous suspensions, solutions and suspensions in oil, emulsions and implants, as well as containers and transdermal devices. In some embodiments, time-consuming genetic construct preparations are recommended. In some embodiments, it is recommended that the genetic construct is allocated in time within 6-144 hours, preferably 12-96 hours, more preferably 18-72 hours.

 In some embodiments of the present invention, the genetic construct is injected using a needleless injection device. Needleless injection devices are particularly preferred for the simultaneous administration of a substance intramuscularly, intradermally and subcutaneously.

 In some embodiments of the present invention, the genetic construct is introduced together with (UVP) using the microparticle particle bombardment technique disclosed by Sanford et al. In US Pat. No. 4,945,050, issued July 31, 1990, which is incorporated herein by reference.

 In some embodiments of the present invention, the genetic construct is introduced as part of a liposome complex with a polynucleotide function enhancing agent.

 In some embodiments of the invention, the individual is subjected to a single vaccination with the creation of a complete, broad immune response. In certain embodiments of the invention, the individual is subjected to a series of vaccinations and a complete, broad immune response is created. According to some embodiments of the invention, at least two, preferably four to five, injections are given over time. The time interval between injections can be from 24 hours to two weeks or more, preferably one week. Or at least two and up to four injections are carried out simultaneously in different areas.

 In some embodiments of the invention, complete vaccination involves injecting a single inoculum containing a genetic construct comprising sequences encoding one or more target epitopes.

 In some embodiments of the invention, comprehensive vaccination involves the injection of two or more different inoculums at different sites. For example, the HIV vaccine of the invention contains two inoculums, each of which includes genetic material encoding different viral proteins. This method of vaccination allows you to enter the most complete set of viral genes without the risk of introducing an infectious viral particle. So, in a vaccinated individual, an immune response can be created to most or all viruses. Inoculation of each inoculant is carried out in different areas, preferably at a certain distance, in order to avoid cells receiving both genetic constructs. For greater security, some genes can be removed or altered with the additional guarantee of preventing the possibility of an infectious viral ensemble. As used herein, the term "pharmaceutical kit" refers collectively to the multiple inoculum used in the present invention. Such kits include separate containers with different inoculum and / or cell stimulating agents. These kits are intended to include a number of inoculums used in the vaccination method.

 The methods of the present invention are applicable in the field of human medicine and veterinary medicine. Accordingly, the present invention relates to genetic vaccination of mammals, birds and fish. The methods of the present invention can be particularly useful when applied to mammals, including humans, cows, sheep, pigs, goats, dogs and cats.

 The following Examples include representative examples of aspects of the present invention. The examples are not intended to limit the scope of the invention, but rather serve the purpose of illustration. In addition, various aspects of the invention are summarized in the following description. However, this description is not intended to limit the scope of the invention, but rather is given to illuminate various aspects of the invention. An ordinary specialist is able to easily identify additional aspects of the invention and its variants.

Example 1
The present invention provides an HIV vaccine useful for direct genetic vaccination. Genetic constructs are given that, when introduced into an individual's cells, are expressed with the production of HIV proteins. In some embodiments, the production of all viral structural proteins in an individual's cells creates a protective immune response that protects against HIV infection. The HIV vaccine of the present invention can be used to vaccinate uninfected individuals against HIV infection or can serve as an immunotherapeutic agent for already infected individuals. The HIV vaccine of the present invention generates an immune response, including CTL, which recognizes and attacks HIV-infected cells and recognizes a wide range of HIV proteins. Thus, uninfected individuals are protected against HIV infection.

 In some of its embodiments, the present invention relates to a method for vaccinating an individual against HIV by administering two inoculums. Such two inoculums include at least two and preferably more than two sets of genes or all genes of the HIV virus. However, inoculums are not supplied together. Accordingly, a complete set of genes does not enter the inoculated cell. The vaccinated individual will receive at least two different and preferably more than two, more preferably a whole series or all of the viral genes.

 Such a strategy increases the likelihood that the genetic material encoding the most effective target proteins will be included in the vaccine, and reduces the likelihood that the virus particle will be able to avoid detection during the immune response, despite structural changes in one or more viral proteins resulting from the mutation the virus. Accordingly, vaccination of an individual with a variety of and, preferably, an almost complete or complete set of genes encoding viral proteins is desirable.

 If a single cell is provided with a complete set of viral genes, it is likely that a complete infectious virus will appear within the cell. Accordingly, the genetic construct of the invention does not contain a complete set of such genes. Moreover, two or more inoculums, each of which has an incomplete set of genes, and all together have up to a complete set of viral genes, are introduced into different cells, preferably located at a certain distance from each other in order to avoid the possibility of a complete set of actions on the vaccinated cell genes. For example, part of the HIV genome can be inserted into the first construct, and the remaining HIV genome can be inserted into the second construct. The first construct is administered to the individual in the form of a genetic vaccine in the muscle tissue of one arm, and the second construct is administered to the individual in the form of a genetic vaccine in the other arm. The individual is exposed to a complete set of viral genes with vaccination thereby essentially from the complete virus, but without the risk of an infectious viral particle.

 For added safety, even if genetic material is delivered by two or more inoculums to spaced parts of an individual’s body, one or more essential genes can be removed or intentionally altered with an even greater guarantee that an infectious viral particle cannot be formed. In such embodiments, an incomplete functional set of viral genes is administered to an individual.

 Additional safety measures are given to the non-overlapping parts of the viral genome in individual genetic constructs, forming correspondingly separate inoculums. This prevents recombination between the two genetic constructs.

 In some embodiments of the present invention, a complete set of structural genes is provided. HIV structural genes include gag, pol and env. These three genes are given in two different DNA or RNA constructs. Accordingly, in one recommended embodiment, gag and pol are in the same DNA or RNA construct, and env in the other. In another recommended embodiment, gag is in one DNA or RNA construct, and pol and env are in another. In another recommended embodiment, gag and env are in the same DNA or RNA construct, and pol is in the other. In some recommended embodiments, constructs containing rev have a splice acceptor upstream of the initiation codon for rev. In certain recommended embodiments, constructs containing gag have a splice donor upstream of the gag translational initiation codon. It is possible that HIV regulatory genes may also be present in any of these combinations. HIV regulatory genes include: vpr, vif, vpu, tat and rev.

 DNA constructs in the recommended embodiment include a promoter, an amplifier, and a polyadenylation signal. The promoter may be selected from the group including HIV DCT, human actin, human myosin, CMV, HRV, Moloni, VOMZHM, human hemoglobin, human muscle creatine and EBV. The enhancer may be selected from the group consisting of human actin, human myosin, CMV, HRV, human hemoglobin, human muscle creatine and EBV. The polyadenylation signal may be selected from the group comprising an DCT polyadenylation signal and an OB40 polyadenylation signal, in particular, among others, a smaller OB40 polyadenylation signal.

 In some embodiments, two inoculum vaccines are administered intramuscularly in spatially separated tissues of an individual, preferably in different parts of the body of the individual, such as, for example, his right and left hands. Each inoculum of the present invention may contain 0.1-10000 μg of DNA. Each inoculum preferably contains 1-500 μg of DNA. More preferably, each inoculum contains 25-250 μg of DNA. Most preferably, each inoculum contains about 100 μg of DNA.

 The inoculum in some embodiments is a sterile isotonic carrier, preferably a phosphate buffered saline or simple saline.

 In some embodiments, a cell proliferative promoting agent, preferably bupivacaine, is injected into the tissues to be vaccinated before the vaccine is administered. Bupivacaine injections can be given up to about 24 hours before vaccination. It is advisable to administer bupivacaine immediately before vaccination. At the injection site, 50 μl to 2 ml of 0.5% bupivacaine-HCl and 0.1% methyl paraben in isotonic NaCl solution are added, preferably 50-1500 μl, more preferably about 1 ml.

 In other embodiments, a cell proliferation promoting agent, preferably bupivacaine, is included in the preparation in conjunction with a genetic construct. Between 50 μl and 2 ml of 0.5% bupivacaine-HCl and 0.1% methylparaben in isotonic NaCl solution, preferably 50-1500 μl, more preferably about 1 ml, are introduced into the place where the vaccine is to be administered.

 Accordingly, two inoculum vaccines are offered in some variants: one inoculum contains a DNA or RNA construct containing two HIV structural genes, the second inoculum includes a DNA or RNA construct containing the third remaining structural gene, so that together the inoculum contains a complete set of HIV structural genes. The structural genes in each DNA construct are operably linked to a promoter, amplifier, and polyadenylation signal. The expression of viral genes can be regulated by the same or different regulatory elements. When vaccinating an individual, two inoculum is administered intramuscularly in different places, preferably in different hands of the individual. In some embodiments of the invention, bupivacaine is first administered at the injection site. In certain embodiments of the invention, bupivacaine is included in a preparation with a genetic construct.

 In some embodiments, the vaccination procedure is repeated at least once, preferably two or three times. Each vaccination is separated by a period of time from 24 hours to two months.

 In some embodiments, the vaccine is administered using a needleless injection device. In certain embodiments, the vaccine is administered hypodermically using a needleless injection device, thereby providing for intramuscular, intradermal and subcutaneous administration at the same time as intermediate administration of the substance.

 Recommended genetic constructs include the following: plasmids and constructs obtained by cloning.

 Two plasmids were constructed: one containing HIV gag / pol, and the other containing HIV env.

 HIV-1 genomic clone pNL43 obtained from Dr. Malcolm Martin, AIDS NCD Program, Research and Reference Reagents (ARRRR), AIDS Division, NIAID, NIH. The clone can be used as a starting product for HIV-1 viral genes intended for genetic constructs. Alternatively, any HIV molecular clone of an infected cell can be modified to a sufficient degree by means of polymerase chain technology, for example: HXB2 clone, MN clone, as well as SP or BAL-1 clone. Clone pNL43 is a construct consisting of HIV-1 proviral DNA plus 3 k.o. the host sequence from the integration site by cloning into plasmid pUC18.

Construction of pNL-puro-env ^- plasmids
This plasmid is designed to express gag / pol. The StuI site within the non-HIV 5'-flanking human DNA from pNL43 is destroyed by partial hydrolysis in the presence of StuI followed by hydrolysis of the free ends in the presence of E. coli polymerase 1. The linear plasmid is filled and then self-ligated, leaving only the StuI site within the HIV genome. This plasmid (pNLDstu) is then hydrolyzed in the presence of blunting enzymes StuI and BsaBI, which remove a fragment of the gp120 coding sequence. In the presence of EcoRI and ClaI from pBABE-puro (Morgenstern, Land, 1990, Nucl. Acids Res. 18 (12), 3587-3596, incorporated herein by reference, a plasmid provided by Dr. Hartmut Land, Imperial Cancer Research Center) isolated OB40 promoter and region of coding for resistance to puromycin (puromycin-acetyltransferase (PAT)). This fragment is blunted and then cloned into StuI / BsaBI-hydrolyzed pNLDstu. A clone with the OB40-puro fragment in the required orientation is selected so that 3'-DCT from HIV can provide poly-A functions for the translation of PAT. The resulting plasmid is designated pNLpuro.

Cloning strategy for deletion of vpr regulatory gene from HIV gag pol vector
The region immediately upstream from the only PflMI site to immediately after the vif termination codon is amplified by PCR using primers that introduce the non-conservative substitution of amino acid (glu ---> val) at amino acid 22 in vpr, the termination codon in vpr a reading frame after amino acid 22 and an EcoRI site located immediately after the new termination codon. This PCR fragment is replaced by a PflMI-EcoRI fragment from pNLpuro or pNL43. Such a substitution leads to a deletion of 122 nucleotides in the open vpr reading frame, thereby eliminating the possibility of reversal characteristic of the even mutation strategy. The resulting plasmid (pNLpuro vpr) encodes the first 21 natural amino acids from vpr plus valine plus all other remaining HIV-1 genes and splice junctions in their native form. A similar deletion strategy also applies to nef, vif and vpu and allows expression of the structural gene while protecting against the formation of a living recombinant virus.

Construction of plasmids designed for expression of the membrane
The DNA segment encoding the HIV-1 HXB2 envelope gene is cloned by polymerase chain reaction (PCR) amplification using lamda-cloned DNA obtained from the AIDS program, studies and reference reagents. The sequences of the 5'- and 3'-primers are as follows: 5'-AGGCGTCTCGAGACAGAGGAGAGCAAGAAATG-3 '(SEQ. N 1) with the introduction of the XhoI site and 5'-TTTCCCTCTAGATAAGCCATCCAATCACAC-3' (SEE. N 2) with the introduction of XbaI Xba on gp160, tat and rev coding region. Gene-specific amplification is carried out using Tag DNA polymerase according to the manufacturer's instructions (Perkin-Elmer Cetus Corp.). The PCR product is treated for thirty minutes at 37 ^° C. with 0.5 μg / ml proteinase K, followed by extraction with a phenol-chloroform mixture and ethanol precipitation. The isolated DNA is then hydrolysed for two hours at 37 ^° C. in the presence of XhoI and XbaI and subjected to agarose gel electrophoresis. The isolated and purified XhoI-XbaI fragment is cloned into a Bluescript plasmid (Strategen Inc., La Jolla, Ka) and then subcloned into the pMAMneoVlue eukaryotic expression vector (Clontech Laboratory, Inc., Ralo Alto, CA). The resulting construct is designated as pM160 (Fig. 1A). Plasmid DNA is purified by CsCl gradient centrifugation. The pM160 DNA construct encodes HIV-1 / HXB2 (Fisher AG et al., (1985) Nature 316, 262-265) membrane bound glycoprotein gp160 under the control of the HRV reinforcing element and VOMGM CDK as a promoter.

Construction of an alternative plasmid for expression of the membrane, called HIV-1 env-rev plasmid
The region encoding the two exons from rev and vpu and the enveloped open reading frames from HIV-1 HXB2 are amplified by PCR and cloned into the pCNDA / neo expression vector (Invitrogen). This plasmid directs the production of the membrane with the participation of the CMV promoter.

Receiving and cleaning
The plasmid in E. coli (DH5 alpha) is grown as follows. LB plus ampicillin agar plates are seeded by hatching the culture of the target plasmid from the frozen total lot. The plates are kept at 37 ^° C. overnight (14-15 hours). A single colony was selected from the plate, which was inoculated with 15 ml of LB medium with peptone and 50 μg / ml ampicillin. The resulting culture was grown at 37 ^o C with shaking (approx. 175 rpm) for 8-10 hours. Indications of OD ₆₀₀ should be at least 1. LB medium with peptone and 50 μg / ml ampicillin (1 L) are inoculated with a culture with an OD value of 1. The prepared culture (1-2 liters) is grown overnight at 37 ^° C. shaking (175 rpm).

 Plasmids grown in E. coli (strain DH5 alpha) are harvested and purified according to the following procedure. General techniques for cell lysis and plasmid purification can be found in: Molecular Cloning. Laboratory Manual, 2nd Edition, J. Sambrook, E.F. Fritsch, T. Maniatis, Could Spring Harbor Press, 1989. The cells are concentrated and washed with glucose-Tris-EDTA buffer (pH 8). Concentrated cells are lysed by the action of lysozyme and briefly treated with 0.2 n. KOH, the subsequent addition of potassium acetate-acetic acid buffer set the pH to 5.5. Insoluble substances are removed by centrifugation. To precipitate the plasmid, 2-propanol is added to the supernatant. The plasmid was redissolved in Tris-EDTA buffer and further purified by extraction with a phenol-chloroform mixture and reprecipitation with 2-propanol.

Endotoxin can be removed using a wide variety of methods, including the following: specific adsorption by immobilized substances, for example polymyxin (Tani et al., Biomater. Aptif. Cells Inimobilization Biotechnol. 20 (2-4), 457-62 (1992); Issekutz, J. Immunol. Methods 61 (3), 275-81 (1983); use of anti-endotoxin monoclonal antibodies, e.g. 8A1 and HA-1A ^TM (Centocor, Malvern, PA, Bogard et al., J. Immunol. 150 (10) 4438-4449 (1993); Rietschel et al. Infect. Immunity, p. 3863 (1993)); use of positively charged depth filters (Hou et al., J. Parenter. Sci. Technol. 44 (4), 204- 9 (July-August 1990)); use of poly (gamma-methi l-L-Glutamate), Hirayama et al., Chem. Pharm. Vull. (Tokyo) 40 (8), 2106-9 (1992)); the use of histidine (Matsumae et al., Biotechnol. Appl. Biochem. 12 (2), 129-40 (1990)); the use of columns and membranes of hydrophobic interaction (Aida et al., J. Immunol. Methods 132 (2), 191-5 (1990); Umeda et al., Biomater. Artif. Cells Artif. Organs 18 (4), 491-7 (1990); Hou et al., Biochem. Biophys. Acta 1073 (1), 149-54 (1991); Sawada et al., J. Hyg. London) 91 (1), 103-14 (1986)); the use of specific resins suitable for the removal of endotoxin, including polystyrene-divinylbenzene or divinylbenzene resins, for example Brownley Polypore resins (Apple Biosystems, Palo Alto, CA); XUS 40323 (Dow Chemical, Midland, MI); HP20, UNR20R (Mitsubishi Kasei, USA); Hamilton PRP-1, PRP-affinity (Hamilton, Renault, HB); Georgy with reversed phases of DVB, Geordi gel of DVB, Polymer Labs PL-gel ^TM (Oltech, Deerfield, IL); Vidak PLx ^TM (Separation Group, Hesperia, KA); other endotoxin-removing materials and methods include; Detoxi-Gel ^TM , removing endotoxin gel (Pierce Chemical, Rockford, IL); Note 206 to use (Pharmacy Biotech, Inc., Pisketaway, NY). See also generally: Sharma, Biotech. Arr. Biochem. 8, 5-22 (1986). Recommended anti-endotoxin monoclonal antibodies bind to the conserved domains of endotoxin and are preferably antibodies to lipid A, the most structurally preserved part of the endotoxin molecule. Such anti-lipid A monoclonal antibodies include: monoclonal antibodies 8A1 with high affinity for mouse IgG and human anti-lipid A IgM (k) monoclonal antibodies HA-1A ^TM . The source of HA-1A ^TM is human E. coli 15 vaccine. As reported, HA-1A ^{TM is} widely cross-reactive with a wide variety of bacterial endotoxins (lipopolysaccharides).

Example 2
In experiments designed to compare the immunogenic response that occurs after genetic vaccination and vaccination with proteins, animal models were created using tumor cells specifically expressing a foreign fractional protein. Three models were created on immunocompetent mice expressing foreign antigens. Three lines of tumor clonal cells are used, the source of which is the Balb / c mouse line. The cell lines are represented by: 1) a lymphoid cell line that does not give significant metastases to other tissues, but forms a palpable tumor, which, apparently, kills the animal within 8-12 weeks; 2) a cell line of murine melanoma with the same ability to form metastases, mostly to the lung, and which, after inoculation with 1 million cells, leads to the development of large palpable tumors in the mouse, which similarly kill the animal within 10-12 weeks; and 3) a murine pulmonary adenocarcinoma cell line that metastases to many tissues and kills the animal for 12 or more weeks. Subclones are selected in which foreign antigens are detected in an unrecognized form. After implantation of a transfected tumor of the mouse of the parent line, unlike most
similar lines of mouse tumors, the animals do not have a protective reaction to existing foreign antigens, and the tumor survives. This tumor subsequently kills the animal with the same phenotype and at the same time intervals as the original non-transfected tumor. The use of such models can determine the immune response generated by genetic vaccination against antigen.

 It was observed that mice vaccinated with genetic vaccines, including a genetic construct, as a result produce in their cells a target protein that creates an immune response, including a strong cytotoxic reaction that completely destroys tumors in which the target protein is found, but without any effect on tumors without the target protein. In mice inoculated with the target protein itself, the resulting immune response was much less effective. The tumor was reduced in size, but due to the absence of a cytotoxic reaction, its destruction did not occur. Nontransfected tumors were used as a control in experiments when comparing the immune response of animals vaccinated with a genetic vaccine, subunit vaccine, and unvaccinated animals. In these experiments, it was clearly shown that a genetic vaccine creates a more effective immune response with a wider spectrum of activity, capable of completely destroying the tumor with the participation of CTLs. Conversely, immunization with an intact target protein creates a more limited, less effective immune response.

Example 3
In another embodiment of the invention, a genetic HIV vaccine is provided. The gp160 viral protein, converted to gp120 and gp41, is the target protein against which the genetic vaccine is obtained. A genetic vaccine contains a DNA construct comprising a DNA sequence encoding gp160 operably linked regulatory elements. When DNA is administered to an individual, the construct of the genetic vaccine is introduced into the cells of the individual, after which gp160 is produced. The immune response generated by the protein has a broad basis and includes the humoral and both types of cellular immune responses. A wide biological reaction provides a higher degree of protection compared to that achieved with the introduction of the protein itself.

 Mice were injected intramuscularly with pM160 (see Example 1), after which an anti-HIV immune response was tested. In the antiserum of animals immunized in this way, an immune response to HIV envelope protein is created, determined by enzymatic immunosorbent analysis (FISA) and immunoprecipitation analysis. The antiserum neutralizes HIV-1 infection and suppresses HIV-1-induced syncytium formation.

 The observed neutralization and anti-syncytial activity may result from the reactivity of the created antibodies to the HIV-1 envelope protein, for example: the V3 loop of gp120, the CD4 binding site and the N-terminal “immunodominant” region of gp41 among others.

 In the genetic immunization method described here, 100 μl of 0.5% bupivacaine-HCl and 0.1% methylparaben in isotonic NaCl solution are injected into the quadriceps muscles of BALB / c mice using a 27-gauge needle. The purpose of the injection is to stimulate the regeneration of muscle cells and facilitate the absorption of the genetic construct. Twenty-four hours later, either 100 μg pM160 or 100 μg pMAMneoBlue was injected into the same injection sites as a control plasmid (Fig. 1A). Mice are revaccinated by injection of the same amount of construct DNA three times with an interval of two weeks in the specified way, but without prior treatment with bupivacaine-HCl.

When immunizing with recombinant gp160, BALB / c mice were initially immunized with 1 μg of glycosylated recombinant (HIV-1 / III ₈ ) gp160 (MicroGenSys Inc.) in complete Freund's adjuvant followed by three re-vaccinations with 1 μg gp160 twice every time weeks. The production of anti-HIV-1 gp160 antibodies is determined by assaying mouse sera for the ability to immunoprecipitate gp160. Immunoprecipitation is carried out using 1 • 10 ⁶ cpm ¹²⁵ J-labeled rgp160 serum of mice and protein-G agarose beads (GIBCO, Inc.) according to the previously described method of Osther K. et al., Hybridoma 10, 673-683 (1991), which is introduced here as a reference. Specific precipitation results are analyzed for 10% VAT-PAGE. Lane 1 corresponds to 1 μl of serum of a pre-sensitized mouse in reaction with ¹²⁵ J-gpl60. Lane 2 corresponds to 1 μl of mouse serum immunized with pM160. Lane 3 corresponds to 1 μl of a 1: 100 dilution of ID6 monoclonal anti-gp120 antibodies (Ugen K.E. et al. (1992) "Creating monoclonal antibodies against the gp120 amino region causing antibody-dependent cell cytotoxicity", Could Spring Harbor Labor) as positive control. The arrow indicates the specifically immunoprecipitated glycoprotein of the ¹²⁵ J-gpl60 membrane.

¹²⁵ J-labeled gpl60 is specifically immunoprecipitated with antiserum from pM160-immunized animals (Fig. 2, lane 2), as well as ID6 monoclonal antibodies used as a positive control (Fig. 2, lane 3). In contrast, the serum of the pre-sensitized mice (FIG. 2, lane 1) showed minimal activity in the same assay.

 Eight out of ten mice immunized with the pM160 construct were positive in terms of reactivity to gpl60 as determined by FISA and the immune response of animals with the highest anti-gp160 titer was subjected to detailed analysis. All four mice immunized with a control vector showed the same negative reactivity to gp160 as determined by FISA, and the serum of one of these mice was used as a control in subsequent experiments.

 HIV neutralizing antibodies have been shown to specifically target certain epitopes of gp120 and gp41, including the V3 loop in gp120 (Goudsmit J. et al., (1988) AIDS 2, 157-164; Putney SD et al., (1989) " Creation of HIV subunit vaccine, Montreal, CD4 binding site near the carboxy terminus of gp120 (Lasky LA et al. (1987) Cell 50, 975-985), as well as the immunodominant gp41 loop directly in the downstream direction from the N-terminal fusion region (Schrier RD et al. (1988) J. Virol. 62, 2531-2536).

To determine whether the anti-gp160 antibodies created in these mice will react with the indicated important regions of envelope glycoproteins, peptides for the BRU / V3 loop, peptides for the MN / V3 loop, peptides for BXB2 / gp41 N-terminus or peptides for HXB2 / CD4, the binding sites are adsorbed onto microtiter plates, after which the specific reactivity of the mouse antiserum is determined by FISA. One μg / ml gp160 or 10 μg / ml of each peptide is coated onto microtiter plates in 0.1 M bicarbonate buffer (pH 9.5), kept overnight at 4 ^° C, blocked with 2% bovine serum albumin in PBS, carried out over one hour at 37 ^o C the reaction with goat anti-mouse IgG conjugated with POH (Fisher) and show 10-30 minutes at room temperature in the dark TMB substrate (Sigma). The results are shown in FIG. 3. The following antisera are shown: (- + -) corresponds to serum of a pre-sensitized mouse, (-x-) corresponds to serum of a mouse immunized with pMAMneoBlue vector, (-o-) corresponds to mouse serum immunized with pM160, (-Δ-) corresponds to mouse serum, immunized with rgp160 protein. In FIG. 3A shows the results obtained using tablets coated with rgp160 protein. In FIG. 3B shows the results obtained using tablets coated with BRU / V3 peptide loops (CNTRKR-IRIQRGPGRAFVTIGK (FOLLOW N 11)). In FIG. 3C shows the results obtained using tablets coated with MN / V peptides (YNKRKRIHIQRGPGRAFYTTKNIIC (SEQ ID NO: 12)) with a QR sequence of HIV-1 / III ₈ underlined type. In FIG. 3D shows the results obtained using tablets coated with HXB2 / CD4 peptides of the binding site (CRIKQFINMWQEVGKAMYAPPISGIRC (FOLLOW N 13)). In FIG. 3E shows the results obtained using tablets coated with the BRU / gp41 peptides of the immunodominant region (RILAVERYTKDQQLLGIWGCSGKLIC (FOLLOW N 14)).

 From FIG. Figure 3 shows that the antiserum from a mouse immunized with the pM160 construct shows a significantly higher reactivity to the BRU and MN / V3 peptides, the CD4 binding site peptide and the immunodominant gp41 peptide than the serum when immunized with the recombinant gp160 protein (rgp160). The antiserum from the rgp160 immunized mouse had a much higher titer relative to rgp160 compared to the antiserum from the pM160 immunization, but lower activity relative to the three specific neutralizing gp160 epitopes analyzed.

To determine whether the antiserum created by immunization with DNA will have antiviral activity, the ability of the antiserum to neutralize HIV-1 infection has been investigated. Infinite HIV-1 / III ₈ virus at 100 TCID _{50 was} incubated with successive dilutions of antiserum before using target cells to infect MT-2 (Montefiori DC (1988) J. Clin. Microbiol. 26, 231-235).

One hundred (100) TCID ₅₀ HIV / III ₈ cell-free viruses were incubated for one hour at 37 ^° C. with successive dilutions of antiserum. After incubation, the pre-treated viruses are applied to plates with the target MT-2 cell line (4 x 10 ⁶ ), incubated for one hour at 37 ^° C, after infection with MT-2, the cells are washed three times and then incubated at 37 ^° C and 5% CO ₂ . Three days later, fusion was quantified by visual counting of the number of syncytium per well in experiments with three repetitions under a phase contrast microscope.

The results obtained are shown in FIG. 4. In FIG. 4A shows the results obtained using serum from a mouse immunized with a vector in comparison with serum when immunized with pM160. Plots of neutralization (V _n / V _o ) versus dilution factor (Nara P. (1989) HIV Research Methods, ed. Aldovini A., Walkter BD, 77-86, M. Stockton Press) are shown in FIG. 4C. Control serum (-x-) was obtained from immunized with pMAMneoBlue mouse vector. Test serum (-o) was obtained from immunized pM160 mice.

The suppression of syncytium is carried out according to the method of Osther K. et al. (1991) Hybridoma 10, 673-683. The H9 / III ₈ cell line was preincubated for thirty minutes at 37 ^° C and 5% CO ₂ with successive dilutions (1: 100, 1: 200 and 1: 400) of antiserum prepared in 96 well plates in a total volume of 50 μl. Three days later, fusion is quantified by visually counting the number of syncytium per well under a phase contrast microscope. FIG. 4D illustrates co-cultivation of target cells with HIV-1 / III ₈ pre-immunized serum cell line. FIG. 4E illustrates the same as FIG. 4D, but the treatment is carried out with serum from the control immunization with the vector. FIG. 4F illustrates the same thing as FIG. 4D, but treatment is carried out with rgp160 immunization serum. FIG. 4G illustrates the same as FIG. 4D, but treatment is carried out with pM160 immunization serum. FIG. 4D-4G show that in this assay, suppression of syncytium becomes apparent at a 1: 200 dilution. MT-2 cells are infected with the acellular HIV-1 / III ₈ virus previously incubated with syncytia, which easily forms in the antiserum from immunization with the vector (Fig. 4A).

In comparison: pre-incubation with serum from an immunized mouse pM160 prevents the formation of syncytium (Fig. 4B). The neutralization kinetics is determined by plotting V _n / V _o versus sequential dilutions (Nara P. (1989) HIV Research Methods, ed. A. Aldovini, Walker BD, 77-86, M. Stockton Press) (Fig. 4C) . The serum from the pM160 immunized mouse exhibits biological neutralizing activity when diluted up to 1: 320, while the control antiserum does not show similar activity.

 In order to determine whether the antiserum from pM160 of an immunized mouse is able to suppress the transmission of the virus transmitted through the membrane by directly fusing the cell with the cell, experiments were carried out to suppress syncytium. The antiserum from the pM160 immunized mouse suppresses HIV-1-induced syncytium formation at a dilution of 1: 200 (FIG. 4G). In contrast, the pre-immune serum, the antiserum from rgp160 of the immunized mouse (Fig. 4F) and the antiserum from the immunized control animals of the vector (Fig. 4E) were not able to suppress the formation of syncytia with the same dilution.

 The observations made in the neutralization experiments (Fig. 4A-C) and inhibition of syncytium formation (Fig. 4D-G) for these sera correlate with the reactivity observations made in FISA (Fig. 3). The antiserum from the pM160 immunized mouse, which showed a high level of binding to neutralizing epitopes, also showed a high level of antiviral activity. Conversely, with a low level of binding to the same epitopes, including the antiserum from rgp160 of an immunized mouse, it has low antiviral activity.

To determine whether the antiserum from pM160 of an immunized mouse is able to inhibit the binding of gp120 to CD4-containing T cells, a direct inhibition assay was performed using fluorocytometry (Chen YH et al. (1992) AIDS 6, 533-539). It was observed that serum from an immunized mouse pM160 construct was able to block the binding of gp120 to CD4-containing T cells. Immune serum at a 1:15 dilution inhibits the binding of FITC-gp120 to CD4 ⁺ SupT1 cells by 22 ± 2% when determined by flow cytometry in duplicate experiments. This indicates that this region of HIV penetration into target cells can also be functionally suppressed by this antiserum. The data obtained correlate with the observed in FISA reactivity of the antiserum to peptides CD4 binding site (Fig. 3C).

 Using a commercially available mouse monoclonal antibody kit for isotype determination (Sigma), immunoglobulin isotype studies were performed. Of the anti-gp160 specific antibodies produced by pM160 immunization, 19% are IgG1, 51% are IgG2, 16% are IgG3, 10% are IgM and 5% are IgA.

 The predominance of IgC isotypes indicates the occurrence of a secondary immune response, which further strengthens the assumption that helper T cells can arise during genetic vaccination.

 The microtiter plates are coated with pM160 and PMAMneoBlue DNA and specific binding is determined by the FISA method using serum from all immunized animals. No significant binding to plasmid DNA was noted. Thus, it is unlikely that using genetic material to inoculate muscle tissue can create a reaction against plasmid DNA.

 The introduction of DNA construct into the muscle of the mouse by injection using a needle can lead to unpredictable results, since this technique does not provide a means of regulating the absorption of DNA by muscle cells. The initiation of DNA construct alone (n ≈ 4) with animals (n ≈ 4) pretreated with bupivacaine was compared. The immune reactions observed in the two groups do not coincide, and the FISA method for these groups yields values of 25% and 75%, respectively, for the responding animals. Increased efficiency can be achieved by using a direct DNA delivery system, for example, particle bombardment (Klein, T.M. et al. (1992) Biotechnology 10, 286-291).

 The facts of neutralization, suppression of syncytium, suppression of CD4-gp120 binding and specific binding to some important areas in gp160 show that the introduction of the DNA construct encoding HIV gp160 membrane-bound glycoprotein directly into the muscles of the animal causes specific humoral reactions and leads to the formation of biologically relevant antiviral antibodies.

 To determine whether the vaccine will be able to create a protective immune response, the above animal model was used. Tumor cells transfect DNA encoding gp160, then confirm the expression of the protein and implant the animal. Control includes nontransfected cell lines.

 Genetically immunized animals are vaccinated with plasmid pM160. The control includes unvaccinated animals, animals vaccinated with vector DNA only, and animals that have been introduced gp160 protein.

 The results show that the immune response of the vaccinated mouse is sufficient to completely remove the transfected tumors without exerting any effect on the non-transfected tumors. Vaccination with gp160 protein leads to a slight decrease in the size of transfected tumors compared to non-transfected tumors, but has no effect on mortality. Unvaccinated animals showed the same mortality for both transfected and non-transfected tumors.

Example 4
The following is a list of constructs that can be used in the methods of the present invention. First post on pBabe vector creation. puro, used as a starting material in the construction of many of the following constructs, appeared in Morgenstenn JP, Land H. (1990) Nucl. Acids Res. 18 (12), 3587-3596, which is incorporated herein by reference. Plasmid pBabe. puro is particularly suitable for the expression of exogenous genes in mammalian cells. The sequences to be expressed are inserted into the cloning sites under the control of the long-term repeat (MAC) of the murine mouse leukemia virus (Mo MuLV) promoter. The plasmid contains a selectable marker for puromycin resistance.

Example 5
Plasmid pBa.Vα3 is a plasmid at 7.8 kb containing 2.7 kb. EcoRI of the genomic fragment encoding the Vα3 region of the T cell receptor containing the L, V and J segments cloned at the EcoRI site of the plasmid pBabe.puro. The target protein originating from the T cell receptor is useful for vaccinating against and treating autoimmune disease transmitted through T cells, as well as clonotypic T cell lymphoma and leukemia.

Example 6
Plasmid pBa. gagpol-vpr is a 9.88 kb plasmid containing gag / pol and vlf genes from HIV / MN cloned into pBabe.puro. The vpr gene has been removed. A plasmid containing the indicated viral genes encoding HIV target proteins is useful for immunization against HIV infection and AIDS and for their treatment. HIV DNA sequence published by: Reiz MS (1992) AIDS Res. Human Retro. B, 1549, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference.

Example 7
Plasmid pM160 is an 11 kb plasmid containing 2.3 kb. PCR fragment encoding HIV-1 / 3B envelope protein and rev / tat genes cloned into pMAMneoBlue. The nef area has been removed. A plasmid containing the indicated viral genes encoding the target proteins is applicable for vaccination against HIV infection and AIDS and for their treatment. DNA sequence HIV-1 / 3B published in the work; Fisher A. (1995) Nature 316, 262, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N K03455, which is indicated here by reference.

Example 8
Plasmid pBa.VL is a 5.4 kb plasmid containing a PCR fragment encoding the VL region of an anti-antibody antibody cloned at pBabe.puro at XbaI and EcoRI sites. The target protein derived from an antibody is an example of a target protein useful for vaccination against B-cell-transmitted autoimmune disease and clonotypic B-cell lymphoma and leukemia. A general technique for cloning functional variable regions from antibodies can be found in: Chaudhary V.K. et al. (1990) Proc. Natl. Acad Sci. US 87, 1066, which is incorporated herein by reference.

Example 9
Plasmid pOspa.B is a 6.84 kb plasmid containing the coding region encoding the OspA and OspB antigens of Borrelia burgdorferi, the spirochete responsible for Lim disease. Antigens are cloned by BamHI and SalI sites of plasmid pBabe. puro. PCR primers used to create OspA and OspB fragments have the following sequences: 5'-GAAGGATCCATGAAAAAATATTTATTGGG-3 '(SEQ. N 3) and 5'-ACTGTCGACTTATTTTAAAGCGTTTTTAAG-3' (SEQ. N 4). See: Williams WV et al. (1992) DNA and Cell. Viol. 11 (3), 207, which is incorporated herein by reference. A plasmid containing the indicated pathogenic genes encoding target proteins is useful for vaccination against Lim disease.

Example 10
Plasmid pBa. RB-G is a 7.1 kb plasmid containing the PCR generated fragment encoding rabies protein C cloned into the BamHI site of pBabe.puro plasmid. The plasmid containing this pathogen gene encoding rabies protein C is useful for rabies vaccination. The DNA sequence is in Genbank under N M32751, which is indicated here by reference. See also: Aniliopis A. et al. (1981) Nature 294, 275, which is incorporated herein by reference.

Example 11
Plasmid pBa.HPV-L1 is a 6.8 kb plasmid containing a fragment created by PCR that encodes the L1 capsid protein of human papillomavirus (HPV), including its strains 16, 18, 31 and 33. The fragment was cloned into BamHI and EcoRI plasmid sites of pBabe.puro. The DNA sequence is placed in the Genebank under N M15781, which is indicated here by reference. See also: Howley P. (1990) "Sections of Virology", Volume 2, ed. Channock RM et al., Chapter 58, 1625, and Shah K., Howley P., "Sections of Virology" (1990), Volume 2, ed. Channock RM et al., Chapter 59, each of which is incorporated herein by reference. The plasmid is applicable for vaccination against HPV infection and in the treatment of cancer.

Example 12
Plasmid pBa.HRV-L2 is a 6.8 kb plasmid containing the fragment created by PCR that encodes the L2 capsid protein of human papilloma virus (HPV), including HPV strains 16, 18, 31 and 33. The fragment was cloned at BamHI and EcoRI sites of plasmid pBabe.puro. The plasmid is applicable for vaccination against HPV infection and in the treatment of cancer. The DNA sequence is placed in the Genebank under N M15781, which is indicated here by reference. See also: Howley P. (1990), "Sections of Virology," vol. 2, ed. Channock et al., Chap. 58, 1625; Shah K., P. Howley (1990) "Sections of Virology", Volume 2, ed. Channock et al., Chapter 59, each of which is incorporated herein by reference.

Example 13
Plasmid pBa. MNp7 is a 5.24 kb plasmid containing the PCR generated fragment encoding the p7 coding region, including the HIV MN gag (core protein) sequence cloned at the BamHI site of pBabe plasmid. puro. Containing these HIV viral genes encoding HIV target proteins, the plasmid is applicable for vaccination against HIV infection and AIDS and for their treatment. See work: Reiz MS (1992) AIDS Res. Human Retro. 8, 1549, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference.

Example 14
Plasmid pGA733-2 is a 6.3 kb plasmid containing tumor surface antigen GA-733-2, cloned from colorectal carcinoma cell line SW948 into pCUM8 vector (Seed B., A. Aruffo (1987) Proc. Natl. Acad. Sci. US 84, 3365, hereby incorporated by reference) in the BstXI site. A tumor-associated target protein is an example of a target protein suitable for vaccinating against and treating a hyperproliferative disease, for example, cancer. The GA733-2 antigen is a targeted antigen against colon cancer. Reported GA733 antigen cited in: Szala S. et al. (1990) Proc. Natl. Acad Sci. US 87, 3542-3546, which is incorporated herein by reference.

Example 15
Plasmid pT4-pMV7 is a 11.15 kb plasmid containing cDNA encoding a human CD4 receptor cloned into a pMV7 vector at the EcoRI site. CD4 target protein is useful for vaccination against T-cell lymphoma and its treatment. Plasmid pT4-pMV7 can be obtained from the AIDS Repository, catalog number 158.

Example 16
Plasmid pDJCA733 is a 5.1 kb plasmid containing the GA733 surface tumor antigen cloned into pBabe.puro at the BamHI site. A tumor-associated target protein is an example of a target protein useful for vaccinating against and for treating a hyperproliferative disease, for example, cancer. GA733 antigen is a targeted antigen against colon cancer.

Example 17
Plasmid pBa.RAS is a 6.8 kb plasmid containing the ras coding region, which is first subcloned from pZIPneoRAS and cloned into pBabe. puro on the BamHI site. The ras target protein is an example of a cytoplasmic signaling molecule. A method for cloning ras is provided by Weinberg (1984) Mol. Cell. Biol. 4, 1577, which is incorporated herein by reference. The ras coding plasmid is useful for vaccination against and for the treatment of hyperproliferative disease, for example, cancer, in particular cancer of the bladder, muscles, lungs, brain and bones.

Example 18
Plasmid pBa.MNp55 is a 6.38 kb plasmid containing the PCR generated fragment encoding the p55 coding region including the HIV MN gag precursor sequence (core protein) cloned into pBabe. puro on the BamHI site. A plasmid containing the indicated HIV viral genes encoding HIV target proteins is useful for vaccination against HIV infection and AIDS and for their treatment. See work: Reiz MS (1992) AIDS Res. Human Retro. 8, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference.

Example 19
Plasmid pBa.MNp24 is a 5.78 kb plasmid containing a PCR generated fragment from a pMN-SFl matrix encoding a p24 coding region including a complete HIV MN gag coding region cloned into pBabe.puro by BamHI and EcoRI sites. A plasmid containing said HIV viral genes encoding HIV target proteins is useful for vaccination against HIV infection and AIDS and for their treatment. See work: Reiz MS (1992) AIDS Res. Human Retro. 8, 1549, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference.

Example 20
Plasmid pBa.MNpl7 is a 5.5 kb plasmid containing the PCR generated fragment encoding the p17 coding region, including the HIV MN gag (core protein) sequence cloned into pBabe.puro at BamHI and EcoRI sites. A plasmid containing said HIV viral genes encoding HIV target proteins is useful for vaccination against HIV infection and AIDS and for their treatment. See work: Reiz MS (1992) AIDS Res. Human Retro. 8, 1549, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference.

Example 21
Plasmid pBa.SIVenv is a 7.8 kb plasmid containing the 2.71 kb PCR generated fragment amplified from a construct containing SIV 239 in pBP322 and cloned into pBabe.puro at BamHI and EcoRI sites. The following primers are used: 5'-GCCAGTTTTGGATCCTTAAAAAAGGCTTGG-3 '(SEQ. N 5) and 5'-TTGTGAGGGACAGAATTCCAATCAGGG-3' (SEQ. N 6). The sequence can be obtained from the AIDS Research Program and reference reagents, catalog N 210.

Example 22
Plasmid pcTSP / ATK.env is an 8.92 kb plasmid containing the PCR generated fragment encoding the entire HTLV encoding region of the HTLV-1 / TSP and / ATK isolates subcloned by the pcDNA1 / neo vector. The following primers are used: 5'-CAGTGATATCCCGGGAGAGACTCCTC-3 '(LAST N 7) and 5'-GAATAGAAGAACTCCTCTAGAATTC-3' (LAST N 8). Messages about the plasmid pcTSP / ATK. env are given in the work: Proc. Natl. Acad Sci. US 85, 3599 (1988), which is incorporated herein by reference, HTLV env target protein is applicable for vaccination against HTLV and T cell lymphoma and for their treatment.

Example 23
Plasmid pBa.MNgpl60 is a 7.9 kb plasmid containing the 2.8 kb PCR generated fragment, amplified from the MNenv containing construct in pSR72 and cloned into pBabe.puro at BamHI and EcoRI sites. The following primers are used: 5'-GCCTTAGGCGGATCCTATGGCAGGAAG-3 '(FOLLOW N 9) and 5'-TAAGATGGGTGGCCATGGTGAAT-3' (FOLLOW N 10). See work: Reiz MS (1992) AIDS Res. Himan Retro. 8, 1459, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference. A plasmid containing said HIV viral genes encoding HIV target proteins is useful for vaccination against HIV and AIDS infection and for their treatment.

Example 24
Plasmid pC. MNp55 is a 11.8 kb plasmid containing a 1.4 kb PCR-generated fragment amplified from the gag region of the MN isolate and cloned into pCEP4 vector. A plasmid containing said HIV viral genes encoding HIV target proteins is useful for vaccination against HIV infection and AIDS and for their treatment. See work: Reiz MS (1992) AIDS Res. Human Retro. 8, 1549, which is incorporated herein by reference. The sequence can be obtained in the Genebank under N M17449, which is indicated here by reference.

Example 25
Plasmid pC.Neu is a 14.2 kb plasmid containing a 3.8 kb DNA fragment, which includes the neu coding region of a human oncogen cut from the LTR-2 / erbB-2 construct and subcloned into the pCEP4 vector. A report on the pC.Neu plasmid is provided in: DiFiore (1987) Science 237, 178, which is incorporated herein by reference. The target protein, peu oncogene, is an example of a growth factor receptor useful as a target protein for vaccination against and for the treatment of a hyperproliferative disease, for example cancer, in particular cancer of the colon, breast, lung and brain.

Example 26
Plasmid pC.RAS is a 11.7 kb plasmid containing a 1.4 kb DNA fragment. including the gas coding region of the oncogen, which is first subcloned from pZIPneoRAS and subcloned into pCEP4 at the BamHI site. Report pC. RAS plasmid is provided by: Weinberg (1984) Mol. Cell. Biol. 4, 1577, which is incorporated herein by reference. The ras target protein is an example of a cytoplasmic signaling molecule. The ras coding plasmid is useful for vaccination against and for the treatment of a hyperproliferative disease, for example, cancer, in particular cancer of the bladder, muscle, lung, brain and bone.

Example 27
Plasmid pNLpuro is a 15 kb plasmid containing gag / pol and the SV40-puro insert. A plasmid containing said HIV viral genes encoding HIV target proteins is useful for vaccination against HIV infection and AIDS and for their treatment.

Example 28
To construct the effectiveness of a genetic vaccine against human CD4, a DNA construct was created in mice. These experiments were created to identify the ability of the vaccine to protect against the antigen of T-cell lymphoma. In T-cell lymphoma, CD4 is a tumor specific antigen. Accordingly, this model shows the ability of a genetic vaccine to protect against T-cell lymphoma. In addition, in these experiments, efficacy against one of the members of the superfamily of immunoglobulin molecules was revealed. CD4 is characterized by high conservation between human and mouse species.

 The applicable animal model is given above. Tumor cells transfect DNA encoding CD4, confirm the expression of protein cells, after which they are implanted into the animal. Control includes nontransfected cell lines. Although animals became immunocompetent, the immune response was not directed against an implanted, CD4-labeled tumor in unvaccinated animals.

 Genetically immunized animals are vaccinated with the plasmid pT4-pMV7 described in Example 15. The control includes unvaccinated animals and animals that were introduced CD4 protein.

 In the genetic immunization procedure described here, 100 μl of 0.5% bupivacaine-HCl and 0.1% methylparaben in isotonic NaCl solution are injected into the quadriceps muscles of BALB / c mice using a No. 27 needle to stimulate muscle cell regeneration and facilitate absorption of the genetic construct. Twenty-four hours later, either 100 μg pT4-pMV7 or 100 μg pMV7 as a control plasmid is injected into the same injection site. Mice are revaccinated by injection of that amount of DNA three times at two-week intervals in the same manner, but without prior administration of bupivacaine-HCl.

 The animals are injected with 1,000,000 CD4-labeled tumor cells. In unvaccinated animals, large tumors form and death occurs within 7-10 weeks. In vaccinated animals, similar deadly tumors did not develop.

 The results show that the immune response in genetically vaccinated mice is sufficient to completely remove transfected tumors, and at the same time does not affect nontransfected tumors. Vaccination with CD4 protein leads to a slight decrease in tumor size in the case of transfected tumors compared to non-transfected tumors, but does not affect mortality. Unvaccinated animals showed the same mortality in both transfected and non-transfected tumors.

Example 29
To construct the effectiveness of the genetic vaccine against human GA733, a DNA construct was created in mice. These experiments were conducted to determine the ability of the vaccine to protect against cancer associated with GA733, such as colon cancer. The applicable animal model is given above. Tumor cells transfect DNA encoding GA733, confirm the ability of the cells to express protein, and then they are implanted into the animal. Control includes nontransfected cell lines.

 In the above method, the immunized animals are vaccinated with the plasmid pGA733-2 described in Example 14. The control includes unvaccinated animals and animals that were injected with GA733 protein.

 The results show that the immune response of genetically vaccinated mice is sufficient to completely remove the transfected, and at the same time does not affect the nontransfected tumors. Vaccination of GA733 protein leads to a slight decrease in the size of the transfected tumor compared to a non-transfected tumor, but does not affect mortality. Unvaccinated animals showed the same mortality in both transfected and non-transfected tumors.

Example 30
To construct the effectiveness of a genetic vaccine against human p185neu, a DNA construct was created. These experiments were conducted to determine the ability of the vaccine to protect against cancer associated with p185 neu, such as breast, lung and brain cancer. The applicable animal model is given above. Tumor cells transfect DNA encoding neu, confirm the expression of the protein by the cells, and then implant the animal. Control includes lines of non-transfected tumors.

 Genetically immunized animals are vaccinated with plasmid pLTR-2 / egbB-2, i.e., a 14.3 kb plasmid containing the coding region of the human neu oncogen, cloned by the above procedure into the LTP-2 vector at the XhoI site. 5'-DCT and 3'-DCT are DCT from Moloni-MuLV. Control includes unvaccinated animals and animals to which pl85neu protein was administered.

 The results obtained show that the immune response of genetically vaccinated mice is sufficient to completely remove transfected tumors, and at the same time does not affect non-transfected tumors. Vaccination with p185 protein leads to a slight decrease in tumor size in the case of a transfected tumor compared to a non-transfected tumor, but does not affect mortality. Unvaccinated animals showed the same mortality in both transfected and non-transfected tumors.

Example 31
To construct the effectiveness of a genetic vaccine against human Ras, a DNA construct was created in mice. These experiments were designed to determine the ability of a vaccine to protect against cancer associated with Ras, such as cancer of the bladder, muscles, lungs, brain and bones. The applicable animal model is given above. Tumor cells transfect DNA encoding Ras, confirm the expression of protein cells, and then they are implanted into the animal. Control includes untransfected tumor lines.

 The above vaccination method for genetically vaccinated animals is vaccinated with the plasmid pBa. RAS described in example 17. The target ras protein is an example of a cytoplasmic signaling molecule. A method for cloning ras is provided by Weinberg (1984) Mol. Cell. Biol. 4, 1577, which is incorporated herein by reference. Control includes unvaccinated animals and animals that have been injected with Ras protein.

Example 32
To construct the effectiveness of a genetic vaccine in mice against the human rabies virus G protein antigen, a DNA construct was created. The applicable animal model is given above. Tumor cells transfect DNA encoding rabies virus G protein, confirm the expression of the protein by the cells, after which they are implanted into the animal.

 Genetically immunized animals are vaccinated with the plasmid pBa.PB-G described in Example 10 using the above vaccination method. The rabies virus target G protein is an example of a pathogenic antigen. The DNA sequence is located in Genebank under N 32751. Control includes unvaccinated animals and animals that were introduced rabies virus G-protein.

Example 33
To construct the effectiveness of a genetic vaccine in mice against the antigen of Lim disease, a DNA construct was created. The applicable animal model is given above. Tumor cells transfect DNA encoding OspA and OspB, confirm the expression of the protein by the cells, after which they are implanted into the animal.

 Genetically immunized animals are vaccinated with the plasmid pOspA.B described in Example 9. Control includes unvaccinated animals and animals to which the OspA and OspB proteins were administered.

Example 34
To identify the effectiveness of a genetic vaccine in mice against a variable region of a human T-cell receptor, a DNA construct was created. These experiments were designed to determine the ability of a vaccine to protect against a protein associated with a cancer associated with a T-cell receptor, for example: T-cell lymphoma and an autoimmune disease transmitted through T-cells. The applicable animal model is given above. Tumor cells transfect DNA encoding Ras, confirm the expression of the protein by the cells, after which they are implanted into the animal.

 Genetically immunized animals are vaccinated with the plasmid pVA.Vα3 described in Example 5 using the above vaccination method.

Example 35
Plasmid pM160 can be used as a starting product for the production of a number of plasmids suitable for the expression of one or more genes from the env part of HIV. Description
design pM160 above. The plasmid is directed to the gp160, tat and rev coding region. The nef gene is missing.

 The promoter that controls the expression of the gp160 / rev / tat gene is VOMZH DKP. The promoter can be removed and replaced by the actin promoter, the myosin promoter, the HIV DCT promoter, and the CMV promoter.

 The gene conferring resistance to ampicillin may be removed or deactivated in some other way. A gene that confers resistance to neomycin can be placed under the control of a bacterial promoter.

 Raus sarcoma virus enhancer can be removed from the plasmid. The HRV booster can be replaced with a muscle creatine booster.

 The gp160 / rev / tat genes overlap and share the same nucleotide sequences in different reading frames. The rev gene can be removed by replacing its initiating codon with another codon. The tat gene can be deleted in a similar way. In each plasmid, with the exception of those that use HIV DCT to control gp160 / rev / tat, one of rev or tat, or both rev and tat, can be deleted. In plasmids using the HIV DCT promoter, the presence of tat is mandatory.

 Table 3 lists the pM160-modified plasmids. Each removed HRV amplifier. In some of them, an amplifier is absent (no), in others there is an amplifier of muscle creatine (UMC). In some of them, the HIV rev gene is present (yes), while in others this gene is absent (no). Some of them have the HIV tat gene (yes), while in others this gene is absent (no).

 Constructs RA-29 - RA-56 are identical to constructs RA-1 - RA-32, respectively, except that in each case the promoter that controls the neomycin gene is a bacterial promoter.

Example 36
Plasmid pNLpuro can be used as a starting product in the creation of several different plasmids expressing HIV gag / pol genes. As indicated above, pNLpuro is designed to express gag / pol. The plasmid pNL-puroΔvpr described above was designed to remove the vpr regulatory gene from the HIV gag pol vector in order to exclude the presence of the necessary regulatory protein from a number of genes introduced during vaccination. In addition to the removal of vpr by ordinary specialists in this field, other changes to the pNLpuro plasmid can be made using standard molecular biology techniques and widely available starting materials.

 Human flanking 5 ′ and 3 ′ sequences from HIV sequences can be removed in several ways. For example, only HIV, OB40-puro and pUC18 sequences can be amplified and reconstructed by PCR.

 From the pNLpuro-based plasmids, a psi region important for virus packaging can be deleted. To remove the psi region of the pNLpuro, the plasmid was cut in the presence of SacI and SpeI. As a result of hydrolysis, the psi region is removed, as well as the 5'-DCT, located in the downstream direction, and part of the gag / pol region in the downstream direction from the psi. To re-insert non-psi sequences by DCT, amplification is carried out with the regeneration of these sequences. Primers have been developed that allow the regeneration of part of the HIV sequence 5 'and 3' to psi without psi regeneration. The primers reformate the SacI site in the 5 ′ portion of the 5′-DCP plasmid. Primers go upstream from the SacI site to the site located directly at the 3'-part of the 5'-end of the psi region with the formation at the 3'-end of the AatI site. Primers starting directly in the 5'-part of the psi region, in addition, create an AatI site, and starting in the 3'-part of the SpeI site, regenerate this site. The fragments created by PCR are hydrolyzed in the presence of SacI, AatI and SpeI and ligated together with the pNLpuro-psi fragment hydrolyzed in the presence of SacI / SpeI. The HIV 5'-DKP promoter can be deleted in the replacement of the Moloni virus promoter, VOMZHM DKP, actin promoter, myosin promoter and CMV promoter.

 The HIV 3'-DCT polyadenylation site can be removed and replaced with OB40 polyadenylation site.

 The gene conferring resistance to ampicillin may be removed or deactivated in some other way.

 Table 4 lists pNLpuro-based constructs in which HIV psi and vpr regions were removed and, in addition, the flanking 5 ′ and 3 ′ regions of the human HIV sequence were removed.

 Constructs LA-17 to LA-32 are identical to constructs LA-1 to LA-16, respectively, except that in each case at least one of the human flanking sequences remains.

Example 37
When creating other constructs for expression of the env gene, this HIV region can be inserted into the commercially available plasmid pCEP4 (Invitrogen). The pCEP4 plasmid is particularly suitable because it contains the origin of the Epstein-Barr virus replication and the coding region of the nuclear antigen EBNA-1, creating high-copy replication without integration. pCEP4 also contains a hygromycin marker under the regulatory control of the thymidine kinase promoter and a polyadenylation site. The HIV env coding region is placed under regulatory control of the CMV promoter and the OB40 polyadenylation site. The coding region of HIV env as a 2.3 kb PCR fragment from HIV / 3B, sequence in the Genebank under N K03455. The pCEP4-derived plasmid (pRA-100) is introduced outside the chromosome, and the plasmid produces the gp160 protein.

Example 38
To obtain other constructs for expression of the env gene, this HIV region can be inserted into the commercially available plasmid pREP4 (Invitrogen). The pREP4 plasmid is particularly suitable because it contains the Epstein-Barr virus origin of replication and the coding region of the EBNA-1 nuclear antigen, which provides high-copy replication without integration. pREP4 also contains a hygromycin marker under the regulatory control of the thymidine kinase promoter and polyadenylation site. The HIV env coding region is placed under regulatory control of the HRV promoter and OB40 of the polyadenylation site. The HIV env coding region was obtained as a 2.3 kb PCR fragment. HIV / 3B form, sequence in Genebank N K03455. The plasmid (pRA-101) obtained on the basis of pREP4 was introduced extrachromosomally, and the plasmid produced the gp160 protein.

Example 39
To create other constructs designed to express gag / pol genes, this HIV region can be inserted into the commercial plasmid pCEP4 (Invitrogen). The pCEP4 plasmid is particularly suitable because it contains the Epstein-Barr virus origin of replication and the coding region of the nuclear antigen EBNA-1, which provides high-copy episomal replication without integration. pCEP4 also contains a hygromycin marker under the regulatory control of the thymidine kinase promoter and the polyadenylation site. The HIV gag / pol coding region is placed under the regulatory control of the CMV promoter and the OB40 polyadenylation site. The HIV gag / pol coding region is derived from HIV MN (sequence in Genebank N M17449), and includes the vif gene. The vpr gene is not included. The plasmid (pLA-100) obtained on the basis of pCEP4, introduced extrachromosomally, produces GAG55, reverse transcriptase, protease and integrase proteins.

Example 40
To create other constructs designed for gag / pol expression, this HIV region can be inserted into the commercial plasmid pREP4 (Invitrogen). The pREP4 plasmid is particularly suitable because it contains the Epstein-Barr virus origin of replication and the coding region of the nuclear antigen EBNA-1, providing high-copy episomal replication without integration. pREP4 also contains a hygromycin marker under regulatory control of the thymidine kinase marker and the polyadenylation site. The HIV gag / pol coding region is placed under regulatory control of the CMB promoter and the OB40 polyadenylation site. The HIV gag / pol coding region is derived from HIV MN (sequence in Genebank N M17449), and includes the vif gene. The vpr gene is not included. The pREP4-derived plasmid (pLA-101) is introduced extrachromosomally, and the plasmid produces GAG55, reverse transcriptase, protease and integrase proteins.

Example 41
The following construct, called pGACPOL.rev here, is applicable for the expression of HIV gag / pol genes.

 The plasmid includes the kanamycin resistance gene and pBR322 origin of DNA replication. Sequences necessary for transcriptional regulation include the cytomegalovirus promoter, Routh sarcoma virus enhancer, and OB40 polyadenylation signal. The sequences contained in pGAGPOL.gev HIV-1 include: a sequence encoding p17, p24 and p15 from gag open reading frame, a sequence encoding a protease, a sequence encoding a reverse transcriptase containing a small deletion, and a sequence encoding an inactive amino termination of integrase from open open reading frames, as well as a coding rev sequence. Each HIV sequence originates from the HIV-1 strain HXB2.

 PGAGPOL.rev provides several levels of security. These include the use of the CMV promoter and the non-retroviral poly (A) region. Moreover, deletion of the ψ sequence limits the packaging of viral RNA. In addition, numerous mutations in reverse transcriptase result in a productively inactive product. Moreover, a large deletion in integrase leads to an inactive product, and a kanamycin resistance marker is used to stabilize bacterial transformants.

 Plasmid pGAGROL.rev is constructed as follows.

 Stage 1. Apply a subclone of part of the HIV-1 (HXB2) genome, cloned in Bluescript (Stratagen). The HIV-1 subclone contains the complete 5'-DCT and the rest of the HIV-1 genome up to nucleotide 5795 (Genbank numbering). HIV-1 sequences are derived from HXB2D (AIDS repository).

 Stage 2. Subject the PCR portion of the gag from the open reading frame of the HXB2D plasmid (AIDS storage). The PCR fragment is cut off in the presence of NotI and SpeI and ligated to the above-described HIV-1 subclone, restricted in the presence of NotI and SpeI.

 Stage 3. Subjected to the PCR complex gag / pol and part of the pol-coding sequences from the HXB2D plasmid (AIDS storage) with primers AFTER. N 15 and AFTER. N 16. Cut off the PCR product in the presence of ClaI and ligate with each other. Ligated fragments were cut in the presence of BclI and SalI and ligated with the plasmid from step 2 hydrolyzed in the presence of BclI and SalI.

 Stage 4. Cut the plasmid from stage 3 in the presence of BspMI and EcoRI and again ligated with adapters formed by annealing the linkers (FOLLOW. N 17 and FOLLOW. N 18).

 Stage 5. The plasmid from stage 4 is cut in the presence of NotI and SalI and ligated with either plasmid 4a or 4b from the description given for pENV (see below). Also cut in the presence of NotI and SatI.

 Stage 6. The plasmid from stage 5 is restricted in the presence of SalI and MluI and ligated with the PCR product obtained by PCR rev with primers AFTER. N 19 and AFTER. N 20.

 Stage 7. The plasmid from stage 6 is cut in the presence of NotI and ligated with the product obtained by PCR of the rev-responsible element in the HXB2D plasmid (AIDS storage) with primers AFTER. N 21 and AFTER. N 22.

 Steps 6 and 7 are optional.

Example 42
The following construct, called pENV here, is applicable for the expression of HIV env genes.

 The plasmid includes the kanamycin resistance gene and pBR322 origin of DNA replication. Sequences necessary for transcriptional regulation include the cytomegalovirus promoter, Routh sarcoma virus enhancer, and OB40 polyadenylation signal. The HIV-1 sequences contained in pENV include a vpu coding sequence, a rev coding sequence, a gp160 coding sequence, a 50% nef coding sequence, a vif coding sequence, and a 13pr carboxy terminating vpr coding sequence. The vpu, rev, gp160, and nef sequences occur in the HIV-1 strain MN. The source of vif and vpr sequences is the HIV-1 strain HXB2.

 PGAGROL.rev provides several levels of security. These include the use of the CMV promoter and the non-retroviral poly (A) region. Moreover, the tat was deleted, and the 50th deletion of nef leads to the “inactive" nef of the product. In addition, vif and vpr are deduced from the normal sequence, and a partial deletion of vpr additionally guarantees an inactive vpr product.

 Plasmid pENV was created as follows.

 Stage 1. The original plasmid pUC18 hydrolyzed in the presence of HindIII and EcoRI. The resulting fragment containing the ColEl origin of replication and the laci gene must be ligated with the EcoRI / HindIII fragment from pMAMneoBlue containing our sarcoma virus enhancer. The resulting plasmid or pMAMneoBlue from Clontech (Palo Alto, CA) can then be hydrolyzed in the presence of HindIII and BglI. Using standard techniques, they are ligated with a fragment containing the kan gene obtained by PCR from a gene block plasmid (Pharmacy).

 Stage 2. If pMAMneoBlue is used as the initial plasmid, it is hydrolyzed in the presence of MluI and EcoRI, the ends are filled with a Klenov fragment from polymerase I and re-ligated.

 Stage 3. Then, the plasmid obtained from pMAMneoBlue or pUC18 is hydrolyzed in the presence of HindIII and ligated to the OB40 poly (A) region and the early splicing region obtained by PCR from pCEP4 (Invitrogen, San Diego, KA) with primers SEQUENCE N 23 and AFTER. N 24.

 Stage 4a. Hydrolyzed in the presence of BamHI and ligated with the CMV promoter obtained by PCR from pCEP4 (Invitrogen, San Diego, CA) with primers AFTER. N 25 and AFTER. N 26.

 Stage 4b. Hydrolyzed in the presence of BamHI and ligated with MoMLV DCT obtained by PCR with primers AFTER. N 27 and AFTER. N 28.

 Stage 5. Hydrolyzed in the presence of NotI and MluI and ligated to the gp160 coding region obtained from pMN-ST1 by PCR with primers AFTER. N 29 and AFTER. N 30.

 Stage 6. Hydrolyzed in the presence of MluI and ligated with sequences encoding vif in its entirety and vpr with a deletion of 13 a. K. at the carboxy terminus, obtained from the HX2BD plasmid (AIDS storage) by PCR with primers AFTER. N 31 and AFTER. N 32.

Example 43
A vaccination system is provided, including:
a pharmaceutical preparation containing about 100 μg pGAGPOL.rev in an isotonic pharmaceutically acceptable solution; and a pharmaceutical preparation containing 100 μg pGAGPOL.rev in an isotonic pharmaceutically acceptable solution. In addition, the vaccination system preferably includes a pharmaceutical preparation containing 1 ml of 0.5% bupivacaine-HCl and 0.1% methyl paraben in an isotonic pharmaceutical carrier.

 In the case of using such a recommended vaccination system, a first series of administrations is first carried out, in which bupivacaine and one of the two pharmaceutical preparations are administered intramuscularly to the individual, preferably into the muscles of the arm and buttocks. Bupivacaine and the second of the two pharmaceutical preparations are administered intramuscularly to the individual in another place, preferably remote from the injection site of the first pharmaceutical preparation, preferably in the other hand or buttock. The sequence of a series of administrations can be divided in time, preferably from 48 hours to two or more weeks between two administrations.

 The vaccination system can be used to vaccinate an individual to protect him from HIV infection or to treat an HIV-infected individual with an immunotherapeutic agent.

Example 44
In some embodiments, the invention relates to a method for vaccinating an individual against HIV by administering a single inoculum. Such an inoculum includes a genetic construct containing at least one, preferably two, more preferably more than two, or a series of HIV virus genes or all structural genes. However, the inoculum does not contain a complete set of all HIV genes. When a complete set of viral genes enters a single cell, it is likely that an entire infectious virus will be assembled within the cell. Accordingly, such a complete set of genes is missing from the genetic construct of the present invention. For safety reasons, one or more essential genes can be deleted or intentionally altered with the additional guarantee of eliminating the formation of an infectious viral particle.

 In some embodiments of the present invention, at least a portion of one, two, or all of the HIV structural genes is provided. HIV structural genes include; gag, pol and env. In the genetic construct, parts of at least one of these genes are given. Accordingly, in some embodiments, at least portions of each of the gag and pol are given in the genetic construct; in certain embodiments, at least a portion of env is given in the genetic construct; in other embodiments, at least a portion of the gag is given in the genetic construct; in certain embodiments, at least a portion of each of pol and env is given in the genetic construct; in certain embodiments, at least a portion of each of the gag and env is given in the genetic construct; in other embodiments, at least a portion of pol is given in the genetic construct. Perhaps the presence of the whole gene. In any of these constructs, the presence of HIV regulatory genes is also possible. HIV regulatory genes include: vpr, vif, nef, vpu, tat and rev.

Example 45
As used herein, the term “expression unit” refers to a nucleic acid sequence containing a promoter operably linked to a coding sequence operably linked to a polyadenylation signal. The coding sequence may encode one or more proteins or fragments thereof. In recommended embodiments, the expression unit is within the plasmid.

 As used herein, the term “HIV expression unit” refers to a nucleic acid sequence containing a promoter operably linked to a coding sequence operably linked to a polyadenylation signal, the coding sequence encoding a peptide having an epitope that is identical or substantially similar to an epitope found in HIV protein. The term “substantially similar epitope” refers to an epitope whose structure is not identical to that of the HIV protein epitope, but which nonetheless produces a cellular or humoral immune response that cross-reacts with the HIV protein. In preferred embodiments, the HIV expression unit comprises a sequence encoding one or more HIV proteins or fragments thereof. In recommended embodiments, the HIV expression unit is within the plasmid.

 In some embodiments of the present invention, a single genetic construct is provided comprising a single HIV expression unit comprising DNA sequences encoding one or more HIV proteins or fragments thereof. As used herein, the term “construct with a single HIV expression unit” refers to a single genetic construct containing a single HIV expression unit. In recommended embodiments, a construct with a single HIV expression unit has the appearance of a plasmid.

 In some embodiments of the present invention, a single genetic construct is provided comprising more than one HIV expression unit, each of which contains DNA sequences encoding one or more HIV proteins or fragments thereof. As used herein, the term “genetic construct with a number of HIV expression units” refers to a single plasmid containing more than one HIV expression unit. In recommended embodiments, the construct with a number of HIV expression units has the appearance of a plasmid.

 In some embodiments of the present invention, a single genetic construct is provided comprising two HIV expression units, each of which contains DNA sequences encoding one or more HIV proteins or fragments thereof. As used herein, the term "genetic construct with two HIV expression units" refers to a single plasmid containing two HIV expression units, i.e. to a genetic construct with a series of HIV expression units containing two HIV expression units. In the case of a genetic construct with two HIV expression units, it is recommended that one of the HIV expression units function in a direction opposite to that of the other HIV expression units. In recommended embodiments, the genetic construct with two HIV expression units is a plasmid.

 In some embodiments of the present invention, an HIV genetic vaccine is provided containing a single genetic construct. A single genetic construct may be a genetic construct with a single HIV expression unit, two HIV expression units, or a genetic construct with a series of HIV expression units that contains more than two HIV expression units.

 In some embodiments of the present invention, an HIV genetic vaccine is provided containing more than one genetic construct in a single inoculum.

 In some embodiments of the present invention, an HIV genetic vaccine is provided containing more than one genetic construct in more than one inoculum. As used herein, the term "multiple inoculum" refers to a genetic vaccine comprising more than one genetic construct, each of which is administered separately. In some embodiments of the present invention, an HIV genetic vaccine is provided containing two genetic constructs. Each genetic construct may be, independently of the other, a genetic construct with a single HIV expression unit, a genetic construct with two HIV expression units, or a genetic construct with a series of HIV expression units containing more than two HIV expression units. In some embodiments, both genetic constructs are a genetic construct with a single HIV expression unit. In separate embodiments, both genetic constructs are represented by genetic constructs with two HIV expression units. In other embodiments, both genetic constructs are represented by genetic constructs with a number of HIV expression units. In some embodiments, one genetic construct is a genetic construct with a single HIV expression unit, and the other is a genetic construct with two HIV expression units. Many variations are quite obvious to the average person, depending on the number of genetic constructs used in the genetic vaccine and the number of HIV expression units that may be present in each genetic construct.

 It is recommended that the genetic constructs of the present invention do not contain specific HIV sequences, in particular those involved in the integration of the HIV genome into the chromosome substance of the cell into which the construct is introduced. It is recommended that the genetic constructs of the present invention do not contain MPCs from HIV. It is also recommended that the genetic constructs of the present invention do not contain the HIV psi site. In addition, it is recommended that the reverse transcriptase gene and integrase gene be deleted. Deletions include the deletion of only certain codons or the replacement of individual codons in order to significantly alter the gene. For example, the initiating codon can be deleted, altered, or shifted outside the frame to form a nucleotide sequence encoding an incomplete or non-functional product.

 It is also recommended that the genetic constructs of the present invention do not contain transcribed tat from HIV. The tat gene overlapping the rev gene can be completely removed by replacing the codons encoding rev with other codons encoding the same amino acid for rev, but not encoding the necessary tat amino acid in the reading frame in which tat is encoded. Or, only certain codons are disabled by substituting, but essentially deleting, the initiating codon for tat and / or by replacing, and essentially, deleting, sufficient codons to form a nucleotide sequence encoding an incomplete or non-functional tat.

 It is recommended that the genetic construct include coding sequences encoding peptides having at least one epitope identical or substantially similar to the epitope of HIV gag, pol, env or rev proteins. Even more preferably, the genetic construct includes coding sequences that encode at least one of the HIV gag, pol, env or rev proteins or fragments thereof. It is recommended that the genetic construct includes coding sequences encoding peptides containing more than one epitope identical or substantially similar to the epitope of HIV gag, pol, env or rev proteins. More preferably, the genetic construct includes sequences encoding more than one HIV gag, pol, env or rev protein, or fragments thereof.

 In some embodiments, the genetic construct includes sequences encoding peptides having at least one epitope that is identical or substantially similar to the epitope of HIV vif, vpr, vpu or nef proteins. In certain embodiments, the genetic construct includes sequences encoding at least one of the HIV vif, vpr, vpu, or nef proteins.

 A genetic construct with a single HIV expression unit may include coding regions of one or more peptides sharing at least one epitope with an HIV protein or fragment thereof in a single expression unit under the regulatory control of a single promoter and polyadenylation signal. It is recommended that genetic constructs encode more than one HIV protein or fragments thereof. A promoter can be any functional promoter in a human cell. As a promoter, the OB40 promoter or the CMV promoter is recommended, preferably the CMV directly early promoter. The polyadenylation signal can be any polyadenylation signal that is functional in a human cell. As the polyadenylation signal, an OB40 polyadenylation signal is recommended, preferably OB40 is a small polyadenylation signal. If there are more than one coding region in a single expression unit, these regions can be directly adjacent to each other or can be separated by non-coding regions. For proper expression, the coding region must have an initiation codon and a termination codon.

 A genetic construct with two HIV expression units may include coding regions of one or more peptides sharing at least one epitope with an HIV protein or fragment thereof in each of two expression units. Each expression unit is under the regulatory control of a single promoter and polyadenylation signal. In some embodiments, it is recommended that genetic constructs encode more than one HIV protein or fragment thereof. In some embodiments, it is recommended that nucleotide sequences encoding gag and pol are present in one expression unit, and nucleotide sequences encoding env and rev are present in another. A promoter may be any functional promoter in a human cell. As the promoter, the OB40 promoter or the CMV promoter is recommended, preferably directly the early CMV promoter. The polyadenylation signal can be represented by any polyadenylation signal functional in a human cell. As the polyadenylation signal, an OB40 polyadenylation signal is recommended, preferably OB40 is a small polyadenylation signal. If there is more than one coding region in the expression unit, these regions can be directly adjacent to each other or can be separated by non-coding regions. For proper expression, coding regions must have an initiation codon and a termination codon.

 According to some embodiments of the present invention, MHC Class II cross-reaction epitope in env is removed and replaced by a similar region from HIV II.

 If the genetic construct contains gag and / or pol, it is usually important that rev. For increased expression of gag and pol together with these genes, in addition to rev, a rev-responsive element may also be provided.

 When generating genetic constructs, it is recommended that the gene used in plasmid 1 env be derived from MN or MN-like isolates, including clinical MN-like isolates, preferably non-syncytially induced clinical isolates, preferably those clinical isolates that are macrophage tropics from early clinical isolates.

 Alternative splicing from a single expression unit can produce a variety of proteins. By providing splicing signals, alternative splicing can be achieved in which different matrices are created that encode different proteins.

Example 46
In FIG. 8 shows four skeletons: A, B, C, and D. FIG. 9 shows the inserts: 1, 2, 3, and 4. Box 1 supports the expression of gag and pol; The rev-responsive element is cloned in a way that retains the HIV splice acceptor. Box 2 is similar to box 1 in that it also supports the expression of gag and pol, but the rev-responsive element is cloned without preserving the HIV splice acceptor. Box 3 supports the expression of gag and pol, includes a deletion of the integrase gene, and does not include a cis-acting rev-responsive element. Box 4 supports expression of rev, vpu, and env. The env gene may have a MHC Class II cross-reaction epitope altered to remove cross-reactivity; the V3 loop can be modified to exclude the possibility of syncytium formation.

 In some embodiments, skeleton A is used with insert 1. Similar constructs possibly contain OB40 origin of replication. Plasmid pAlori + is a skeleton A with insert 1 and OB40 origin of replication. Plasmid pAlori- is a skeleton A with insert 1 and without OB40 of origin replication. In addition, both pAlori + and pAlori- can include integrase, which leads to plasmids pAlori + int + and pAlori-int +, respectively. The plasmids pAlori +, pAlori-, pAlori + int +, and pAlori-int + can be further modified by functional deletion of the reverse transcriptase (RT) gene to form pAlori + RT-, pAlori-RT-, pAlori + int + RT, and pAlori-int + RT, respectively -.

 In some embodiments, skeleton A is used with insert 2. Similar constructs may contain OB40 origin of replication. Plasmid pA2ori + consists of skeleton A with insert 2 and OB40 of the origin of replication. Plasmid pA2ori- consists of skeleton A with insert 2, but without the OB40 origin of replication. In addition, both pA2ori + and pA2ori- can include integrase, which leads to plasmids pA2ori + int + and pA2ori-int +, respectively. The plasmids pA2ori +, pA2ori-, pA2ori + int + and pA2ori-int + can be further modified by functional deletion of the reverse transcriptase (RT) gene to form pA2ori + RT-, pA2ori-RT-, pA2ori + int + RT- and pA2ori-int + RT, respectively -.

 In some embodiments, the skeleton B is used with insert 1. Similar constructs possibly contain OB40 origin of replication. Plasmid pBlori + consists of skeleton B with insert 1 and OB40 origin of replication. Plasmid pBlori- consists of skeleton B with insert 1 but without OB40 origin of replication. In addition, both plasmid pBlori + and pBlori- can include integrase, which leads to plasmids pBlori + int + and pBlori-int +, respectively. Plasmids pBlori +, pBlori-, pBlori + int + and pBlori-int + can be further modified by functional deletion of the reverse transcriptase (RT) gene to form pBlori + RT-, pBlori-RT-, pBlori + int + RT- and pBlori-int + RT, respectively -.

 In some embodiments, skeleton B is used with insert 2. Such constructs possibly contain OB40 origin of replication. Plasmid pB2ori + consists of skeleton B with insert 2 and OB40 of the origin of replication. Plasmid pB2ori- consists of skeleton B with insert 2, but without the OB40 origin of replication. In addition, both pB2ori + and pB2ori- can include integrase, which leads to plasmids pB2ori + int + and pB2ori-int +, respectively. Plasmids pB2ori +, pB2ori-, pB2ori + int + and pB2ori-int + can be further modified with functional deletion of the reverse transcriptase (RT) gene to form pB2ori + RT-, pB2ori-RT-, pB2ori + int + RT- and pB2ori-int +, respectively RT-.

 In some embodiments, skeleton A minus rev is used with insert 3. Such constructs may possibly contain OB40 origin of replication. Plasmid pA / r-3ori + has skeleton A with insert 3 and OB40 origin of replication. Plasmid pA / r-3ori- has a skeleton A minus rev with insert 3 and without OB40 origin of replication. In addition, both pA / r-3ori + and pA / r-3ori- can include integrase, which leads to plasmids pA / r-3ori + int + and pA / r-3ori-int +, respectively. Plasmids pA / r-3ori +, pA / r-3ori-, pA / r-3ori + int + and pA / r-3ori-int + can be further modified by functional deletion of the gene for reverse transcriptase (RT) with the formation respectively pA / r- 3ori + RT-, pA / r-3ori-RT-, pA / r-3ori + int + RT- and pA / r-3ori-int + RT-.

 In some embodiments, skeleton C is used with insert 1. Such constructs possibly contain OB40 origin of replication. Plasmid pClori + has skeleton C with insert 1 and OB40 origin of replication. Plasmid pClori- has skeleton C with insert 1 but without OB40 origin of replication. In addition, both pClori + and pClori- can include integrase, resulting in plasmids pClori + int + and pClori-int +, respectively. The plasmids pClori +, pClori-, pClori + int + and pClori-int + can be further modified by functional deletion of the reverse transcriptase (RT) gene to form pClori + RT-, pClori-RT-, pClori + int + RT- and pClori-int + RT, respectively -.

 In some embodiments, skeleton C is used with insert 2. Such constructs possibly contain OB40 origin of replication. Plasmid pC2ori + has skeleton C with insert 2 and OB40 origin of replication. Plasmid pC2ori- has skeleton C with insert 2, but without OB40, origin of replication. In addition, both pC2ori + and pC2ori- can include integrase, which leads to plasmids pC2ori + int + and pC2ori-int +, respectively. The plasmids pC2ori +, pC2ori-, pC2ori + int + and pC2ori-int + can be further modified by functional deletion of the reverse transcriptase (RT) gene to form pC2ori + RT-, pC2ori-RT-, pC2ori + int + RT- and pC2ori-int + RT, respectively -.

 In some embodiments, skeleton C is used with insert 3. Such constructs possibly contain OB40 origin of replication. Plasmid pC3ori + has skeleton C with insert 3 and OB40 origin of replication. Plasmid pC3ori- has skeleton C with insert 3, but without the OB40 origin of replication. In addition, both pC3ori + and pC3ori- can include integrase, which leads to plasmids pC3ori + int + and pC3ori-int +, respectively. Plasmids pC3ori +, pC3ori-, pC3ori + int + and pC3ori-int + can be further modified by functional deletion of the reverse transcriptase (RT) gene to form pC3ori + RT-, pC3ori-RT-, pC3ori + int + RT- and pC3ori-int + RT, respectively -.

 In some embodiments, skeleton D is used with insert 4. Such constructs possibly contain OB40 origin of replication. Plasmid pD4ori + has a skeleton D with insert 4 and OB40 origin of replication. Plasmid pD4ori- has a skeleton D with insert 4, but without OB40 origin of replication.

Example 47
In some embodiments, a single expression unit / single inoculum is given as a genetic vaccine. A vaccine is a genetic construct that includes a sequence encoding a peptide that comprises at least one epitope that is identical or substantially similar to the epitopes of HIV proteins. The coding sequence is under regulatory control of the directly early promoter of CMV and the small signal polyadenylation OB40.

 In some embodiments, a single expression unit / single inoculum is given as a genetic vaccine. A vaccine is a genetic construct comprising a sequence encoding at least one HIV protein or fragment thereof. The coding sequence is under regulatory control of the directly early promoter of CMV and a small signal polyadenylation OB40. The HIV protein is selected from the group consisting of: gag, pol, env and rev. In some recommended embodiments, the genetic vaccine is a genetic construct comprising a sequence encoding at least two HIV proteins or fragments thereof selected from the group consisting of: gag, pol, env and rev, or fragments thereof. In certain recommended embodiments, a genetic vaccine is a genetic construct comprising a sequence encoding at least three HIV proteins or fragments thereof selected into groups comprising: gag, pol, env and rev, or fragments thereof. In some recommended embodiments, a genetic vaccine is provided that is a genetic construct comprising a sequence encoding gag, pol, env and rev, or fragments thereof.

 In some embodiments, a double expression unit / single inoculum is given as a genetic vaccine. A vaccine is a genetic construct comprising two expression units, each of which contains sequences encoding a peptide having at least one epitope that is identical or substantially similar to the epitopes of HIV proteins. The coding sequence is under the control of CMV directly from the early promoter of CMV and OB40 of a small polyadenylation signal.

 In some embodiments, a double expression unit / single inoculum is given as a genetic vaccine. A vaccine is a genetic construct comprising two expression units, each of which contains a sequence encoding at least one HIV protein or fragment thereof. Each expression unit contains a coding sequence under the regulatory control of the CMV of the immediate early promoter and OB40 of the small polyadenylation signal. HIV proteins are selected from the group consisting of: gag, pol, env and rev. In some recommended embodiments, a genetic vaccine is provided, which is a genetic construct comprising two expression units, at least one of which contains a sequence encoding at least two HIV proteins or fragments thereof selected from the group consisting of: gag, rol, env and rev or fragments thereof, and the other contains at least one HIV protein or fragments thereof selected from the group consisting of gag, pol, env and rev or fragments thereof. In certain recommended embodiments, a genetic vaccine is provided, which is a genetic construct comprising two expression units, at least one of which contains a sequence encoding at least three HIV proteins or fragments thereof selected from the group consisting of: gag, pol, env and rev or fragments thereof, and the other contains at least one HIV protein or fragments thereof, selected from the group consisting of: gag, pol, env and rev or fragments thereof. In other recommended embodiments, a genetic vaccine is provided, which is a genetic construct consisting of two expression units and comprising a sequence encoding gag, pol, env and rev, or fragments thereof.

Example 48
A genetic construct (plasmid pCMN160Δ16) has been created for use in an anti-HIV pharmaceutical kit or pharmaceutical preparation. pCMN160Δ16 is constructed as follows.

 Stage 1. To obtain the genomic DNA of the PCR fragment from HIV / MN, primers are used AFTER. N 35 and AFTER. N 34.

 Stage 2. To obtain a PCR fragment from HIV / MN genomic DNA, primers are used AFTER. N 33 and AFTER. N 36.

 Stage 3. Primers LAST. N 35 and AFTER. N 36 is mixed with 2 μl of the reaction product of steps 1 and 2.

 Step 4. The reaction product of step 3 is cut with NotI and MluI and inserted into the skeleton A described in Example 46 and cut with NotI and Mlul.

 As a result, plasmid pCMN160Δ16 is created containing, as an insert into skeleton A, a coding region encoding an ENV protein of the MN strain with a rev region and half nef having changes in the HLA-DB region of HIV-2.

Example 49
Plasmid pGAGPOL. rev2 is prepared as follows. First, the skeleton is prepared. Then form an insert with HIV gag and pol genes, which is inserted into the skeleton.

 The skeleton is obtained as follows.

 Stage 1. The plasmid pMAMneo (Clontech) is hydrolyzed in the presence of BglI. Fill with a maple fragment of polymerase I. HindIII is cut. A fragment of 1763 bp was isolated on a gel.

Stage 2. The Kan ^R gene is amplified from plasmid pJ4Ωkan ⁺ (the kanamycin resistance gene, obtained from Pharmacy Inc., cloned into pJ4Ω, received as a gift from the Imperial Cancer Research Foundation, UK; reports on the construction of pJ4Ω first appeared in: Morgenstern JP, N. Land, Nucl. Acids Res. 18 (4), 1068, which is incorporated herein by reference, with the oligonucleotides SEQ ID NO: 37 and SEQ ID NO: 38. The PCR product is blunted, HindIII is cut, the PCR fragment is gel purified.

Stage 3. The vector skeleton formed from pMAMneo and described for stage 1 is ligated with a PCR product encoding the Kan ^R gene and described for stage 2. A plasmid containing the Kan ^R gene and bacterial origin of replication is isolated.

 Stage 4. The resulting plasmid is hydrolyzed in the presence of MluI, filled with a Klenov fragment from DNA polymerase I. Ligated with a SacII linker (New England Biolabs).

 Step 5. The plasmid obtained in Step 4 is hydrolyzed in the presence of AseI and SspI.

Stage 6. Subjected to PCR part of the Kan ^R gene from the plasmid described for stage 3, using primers AFTER. N 39 and AFTER. 40. Cut the NDP product SspI and AseI.

 Step 7. The largest fragment obtained in step 5 is ligated with the PCR product obtained in step 6.

Stage 8. Cut the ligation product / plasmid,
obtained in stage 7, in the presence of HindIII. Fill with a maple fragment of DNA polymerase I.

 Stage 9. In the presence of SelI, pCEP4 (Invitrogen) was cut to isolate the DNA fragment containing the CMV promoter, polylinker and poly (A) OB40 region. The resulting fragment is purified and blunted by the Klenov fragment of DNA polymerase I.

Step 10. The plasmid obtained in step 8 is ligated with the fragment obtained in step 9. A plasmid containing bacterial origin of replication, Kan ^R gene, HRV amplifier, CMV promoter, polylinker and OB40 poly (A) region is isolated.

 Step 11. The plasmid obtained in step 10 is cut with BamHI and NbeI.

 Stage 12. Annealed oligonucleotides AFTER. N 41 and AFTER. N 42.

 Step 13. The plasmid obtained in step 10 is ligated with the oligonucleotides obtained in step 12. A plasmid containing the adapter from step 12 is isolated.

 Step 14. The plasmid obtained in step 13 is hydrolyzed in the presence of SalI and MluI.

 Step 15. By PCR, the rev open reading frame is amplified using BBG35 (RD Systems Inc. Minneapolis, MN, contains the rev coding region of the HIV strain HX3B in pUC19) as the template and primers AFTER. N 43 and AFTER. N 44. The PCR product is hydrolyzed in the presence of SalI and MluI.

 Step 16. The plasmid obtained in step 14 is ligated with the PCR product obtained in step 15. A plasmid containing the coding region rev. Is isolated.

 Getting the gag / pol insert.

 Stage 1. Subclone part of the HIV-1 (HXB2) genome cloned in Bluescript (Stratagen). The HIV-1 subclone contains the complete 5'-DCP and the remainder of the HIV-1 genome to nucleotide 5795 (Genebank Numbering), cloned into the XbaI and SalI Blue-script sites. From the HXB2D plasmid (AIDS storage), HIV-1 sequences are obtained.

 Stage 2. PCR is subjected to part of the gag coding region from the open reading frame of the plasmid described in stage 1 (subclone of the HIV-1 HXB2 part of the genome cloned in Blue-script) using primers AFTER. N 45 and AFTER. N 46.

 Step 3. The plasmid described in Step 1 (a subclone of the HIV-1 HXB2 part of the Bluescript cloned genome) is hydrolyzed in the presence of EcoRI. The plasmid containing the pBluescript skeleton, 5 'HIV-1 DCT, the gag coding region and part of the pol coding region are purified and religion is purified.

 Step 4. The plasmid obtained in Step 3 is cut off in the presence of NotI and SpeI and ligated to the fragment described for Step 2 by PCR after hydrolysis in the presence of NotI and SpeI. A plasmid was isolated containing the PCR fragment instead of the NotI / SpeI fragment, including 5 'HIV-1 DCT.

 Step 5. The plasmid obtained in Step 4 is hydrolyzed in the presence of EcoRI and SalI.

 Stage 6. Annealed oligonucleotides AFTER. N 47 and AFTER. N 48.

 Step 7. The plasmid obtained in step 5 is ligated with the adapter obtained in step 6. A plasmid containing an adapter cloned at EcoRI / SalI sites is isolated.

 Step 8. The plasmid obtained in Step 7 is hydrolyzed in the presence of NdeI and EcoRI.

 Stage 9. The PCR method amplifies the Rev-responding element (RRE) from a plasmid containing the RRE sequences from the HIV-1 strain HXB2, using primers AFTER. N 49 and AFTER. N 50. The PCR product is hydrolyzed in the presence of NdeI and EcoRI.

 Step 10. The plasmid obtained in step 8 is ligated with the PCR product obtained in step 9. A plasmid containing an insert with an RRE sequence is isolated.

 Step 11. The plasmid obtained in step 10 is hydrolyzed in the presence of NotI and SalI and a fragment containing the gag coding region, the modified pol coding region and the RRE sequence is isolated.

 Stage 12. According to the above procedure for obtaining the skeleton in the presence of NotI and SalI, the plasmid obtained in stage 16 is hydrolyzed.

 Step 13. The plasmid obtained in step 12 is ligated with the insert obtained in step 11. Plasmids containing the insert including the gag coding region, the modified pol coding region, and the RRE sequence are isolated.

 Step 14. The plasmid obtained in step 13 is hydrolyzed in the presence of XbaI and NheI, the ends are blunt and re-ligated.

 Stage 15. Obtaining at stage 14, the plasmid is hydrolyzed in the presence of KpnI and the largest fragment is isolated.

 Stage 16. Annealed oligonucleotides AFTER. N 51 and AFTER. N 52.

 Step 17. The purified plasmid fragment obtained in step 15 is ligated to the adapter obtained in step 16. A plasmid containing an adapter inserted into the KpnI site of the plasmid obtained in step 15 is isolated.

Example 50. Genetic vaccination with regulatory protein genes
Difficulties in the fight against HIV are partly due to the extreme variability of the virus and its ability to rapidly mutate into new forms. There are significant changes in the protein sequence not only in HIV isolates found among the global population, but the virus mutates so quickly that every HIV-infected individual actually carries a number of HIV microvariants. Such HIV isolates are characterized by differences in replication efficiency, tropism, neutralization tendency, and drug resistance. With the advent of drug-resistant mutants, the benefits of drug therapy disappear. In the case of AZT, drug resistance usually occurs during the first year of treatment. Such a constant occurrence of “escaping” mutants may partially affect the ability of HIV to ultimately suppress host defense after a long period during which the virus is apparently under control.

 There are reports of such mutational drift in various parts of the gp120 envelope glycoprotein, including the principal neutralizing domains of the V3 loop, as well as in the proteins of the HIV core. HIV regulatory proteins are much more conservative than structural proteins, and, in addition, are characterized by less mutational drift in time. Thus, regulatory proteins appear to be an attractive target for antiviral attacks.

 HIV is characterized by a noticeable temporal regulation of the expression of regulatory proteins compared to structural ones. In the early stages of viral replication, mRNA encoding Tat, Rev, and Nef regulatory proteins predominates, while in the later stages there is an increased expression of mRNA encoding structural proteins, including Gag, Pol, and Env precursors and many auxiliary proteins. This shift from early to late stages stops when a certain level of Rev protein is reached. The prevalence of Tat, Rev, and Nef in the early stages of the viral cycle also makes these proteins favorable targets for antiviral attack. This is especially true for tat and rev, which play an absolutely important role in the transcriptional and post-transcriptional regulation of HIV gene expression, and which prevail in the early stages of the viral replication cycle before transcription of viral structural proteins and the production of infectious viral particles.

 Unlike tat and rev, which uniquely play a significant role in HIV replication, other proteins, for example: nef, vpr, vif and vpu, are sometimes called “auxiliary” proteins. Their functions are less well understood, and the extent to which viral replication can be impaired by the loss of a particular function varies widely and may depend on the infected host cell. Nevertheless, the strong degree of conservatism of such functions in a wide variety of HIV isolates, as well as in other primitive immunodeficiency viruses, suggests the importance of such “auxiliary” functions in the process of natural infection (see generally: Terwilliger EF (1992) AIDS Research Peviews 2, 3 -27, ed. WC Koff, F. Wong-Staal, RC Kennedy, New York, Marcel Dekker, Inc.). In fact, primitive recombinant viruses with a deletion of vpr, nef or vif are non-pathogenic in vivo, further emphasizing the importance of these auxiliary genes in the life cycle of the virus.

 There is some evidence that a protective immune response against HIV at a higher level can be achieved by selecting several regulatory and / or enzymatic proteins, but not the entire set of HIV genes. Accordingly, a focused vaccination strategy desirably includes genetic vaccination using sequences encoding one or more regulatory, non-structural HIV proteins, including tat, rev, vpr, nef, vpu or vif. Only vpr has been found to be associated with viral particles, while other regulatory proteins, including tat, rev, nef, vif and vpu, are not associated with the virion.

 In some variants of genetic vaccination against HIV using regulatory genes, one of the tat, rev, nef, vif or vpu genes or several such genes are inserted into the skeleton described in Example 46. The use of tat and / or rev is recommended. In some embodiments, tat or rev. Is inserted into the skeleton A described in Example 46. In some embodiments, nef, vpr, vif and vpu are used (the list follows in order of decreasing acceptability as goals). The use of more than one regulatory gene is recommended, including: tat and rev; tat, rev and nef; tat, rev, nef and vpr; tat, rev, nef, vpr and vif; tat, rev, nef, vpr, vif and vpu, as well as combinations thereof and possibly additional regulatory genes such as tev.

 Tat protein is a transactivator of DCP-directed gene expression. Protein is absolutely essential for HIV replication. Tat. produced in the early stages of the viral replication cycle, and functional Tat is required for expression of Gag, Pol, Env and Vpr. The predominant form of Tat is a 86 amino acid protein derived from two-exon mRNA. Amino-terminal 58 amino acids are sufficient for transactivation, albeit with reduced activity. Tat influences a cis-acting sequence called tar, providing a significant increase in DCT-driven gene expression. The effect of Tat can be carried out partly by increasing the synthesis of RNA and partly by increasing the amount of protein synthesized through the RNA transcript. Until recently, it was believed that Tat only affects HIV-1 DCT. However, there have also been reports of Tat-activated expression from the late promoter of the JC virus. As an exogenous factor, Tat can also stimulate cell proliferation and can contribute to promoting the growth of Kaposi’s sarcoma in HIV-infected individuals. Due to this potentially dangerous effect on HIV-infected and uninfected individuals, the recommended tat constructs used for genetic vaccination are modified with expression of only non-functional Tat. Mutations capable of deactivating Tat or Rev can also act as transdominant mutations with potential deactivation as a result of any functional Tat produced in the body of HIV-infected individuals.

 Rev is the second HIV regulatory protein necessary for viral replication. Rev is a 19 kD protein (116 amino acids) expressed from two coding exons in a wide range of spliced mRNAs. Two distinct domains were identified: the main region involved in binding to RRE (Rev-responsive element) containing transcripts, and the “activation” domain, which induces nuclear export of such transcripts as a result of binding. During natural viral infection, Rev is required for the expression of HIV structural proteins Gag, Pol and Env, as well as Vpr.

 Vpr, a protein of 15 kDa (96 amino acids), is found in most HIV-1 strains, although the Vpr open reading frame is intensively truncated in many viral strains during intensive passage in cell culture. A Vpr open reading frame is also present in HIV-2 and most isolates. Vpr is the first retroviral regulatory protein to be found to be associated with HIV viral particles. Its presence in HIV virions suggests the possibility of protein participation in some early stages of the viral replication cycle. Vpr accelerates HIV replication, especially in the early stages of infection. Vpr increases approximately three times the level of expression of reporter genes associated with HIV DC. Moreover, Vpr and Tat apparently act synergistically on DCT-linked genes. Vpr can be isolated from the serum of HIV-infected individuals, and, apparently, increases the ability of the virus to infect new cells. Vpr has also been found to inhibit cell proliferation and induce cell differentiation (Levy DM et al. Cell (1993) 72, 1-20), a discovery that may be important in view of reports that primary monocytes / macrophages are infectious in vitro only undergoing differentiation (Schuitemaker N. et al., J. Clin. Invest. 89, 1154-1160 (1992)). Identical cells that are unable to support HIV replication can be deregulated by Vpr. For example, Vpr may be responsible for the loss of) muscle tissue often seen in AIDS patients. Due to the potentially dangerous activity of Vpr, genetic vaccination should preferably be carried out with a modified vpr construct expressing a non-functional Vpr protein.

 Nef (also called 3'orf in older sources) is a 25-27 kD protein. It has been suggested that Nef may be involved in the upregulation of CD4 + T lymphocytes. In addition, Nef can participate in cellular signals. Nef appears to be important for establishing HIV infection in vivo. Nef-specific CTLs are believed to be important for regulating HIV infection in vivo.

 Vif is a 23 kD cytoplasmic protein called the "virus infectivity factor". Although Vif-defective mutants are not compromised with cell-to-cell transmission, such mutants are characterized by a marked decrease in the ability to infect many CD4 + cell lines. In the absence of Vif, a decrease in wetting and a decrease in infectivity are observed. In primitive experiments, Vif mutants with deletion show a severely reduced ability to create an infection in vivo. These studies confirm the clinical role of Vif in virus replication in the host.

 Vpu is a 15-20 kD protein (81 amino acids). Although Vpu (+) and Vpu (-) viruses produce the same amount of protein, the latter are characterized by increased intracellular accumulation of viral proteins while reducing extracellular virus. This implies the participation of Vpu in the assembly and / or isolation of viral particles.

 Simple retroviruses, for example: mouse-to-bird viruses do not have proteins similar to HIV-1, HIV-2 and SIV regulatory proteins. In such animals, a retroviral infection tends to self-limit with virus clearance and reduced pathogenicity. Similarly, HTLV-1, which includes only Tax (acting almost like Tat and, moreover, showing vpr-like activity) and Rex (acting almost like Rev), undergoes clearance in many individuals. Genetic vaccination with regulatory genes is thought to be appropriate not only for HIV, but also for EBV (X gene product), HCV and HTLV-I (Tax) and (Rex). In all of these viruses, regulatory genes are believed to play a crucial role in the life cycle of the virus and the creation of infection.

Example 51. Construction of HIV-1 regulatory plasmids (pREC)
The pREC plasmid was constructed in stages, and each intermediate could be tested for protein expression before proceeding. An expression vector supporting expression of tat and rev is constructed in two stages. First, the amplification product containing the 5 'NheI site, the HIV-1 main splice donor region, the main part of the tat coding region, the coding region encoding the amino terminal region of the rev protein, and the AvaII site are amplified from a synthetic matrix. Such a synthetic matrix was created using published HXB2 sequences of the HIV-1 strain obtained from the GenBank database, and the sequences were changed by mutations in the tat protein of cysteine residues at positions 22 and 30. Such mutations have been shown to render tat non-functional (Kuppuswany et al. (1989 ) Hucleic Acids Research 17 (9), 3551-3561).

The PCR product is ligated into a vector hydrolyzed in the presence of NheI and AvaII and containing the kanamycin resistance gene and the pBP322 origin of replication. In addition, this plasmid contains a cytomegalovirus promoter, Routh sarcoma virus enhancer, rev coding region, and OB40 polyadenylation signal. The rev sequence present in the plasmid is derived from the proviral clone HIV-1 III ₈ . As a result, an expression vector is created containing the complete but mutated tat coding region and the full rev coding region.

 The next step is to create a PCR product containing the AvaII site at its 5'-end, a mutation at the amino acid position 81 rev, about 30% of the coding region rev, about 30% of the coding region nef and MluI site and 3'-end. Replacing the amino acid at position 81, as shown, leads to the removal of rev functions, as a result of which the resulting plasmid will produce a non-functional rev protein (Bogard N., Greene W.C. (1993) J. Virol. 67 (5), 2496-2502). It is believed that a major deletion in the nef coding region will lead to the production of a non-functional nef protein. The 5 ′ AvaII site and the mutation at the amino acid at position 81 rev of the protein are introduced into the 5 ′ PCR primer complementary to the rev coding region containing both the AvaII site and the nucleotide encoding amino acid 81. Using the 3 ′ PCR primer, a termination codon is introduced that causes the termination of Nef at the amino acid at position 63, and the 3 'MluI cloning coding site. The matrix for such PCR amplification is a plasmid or synthetic matrix containing the coding region rev and nef from the MN strain of HIV-1. The resulting PCR product is hydrolyzed in the presence of AvaII and MluI and used to replace a smaller AvaII-MluI fragment formed after hydrolysis in the presence of AvaII and MluI described in the previous paragraph of the tat-rev plasmid.

 An introduction to one of the two sites of this plasmid vpr is possible. In one approach, vpr can be amplified by applying a 5 ′ PCR primer containing the upstream MluI site in a sequence spanning the vpr translation initiation codon, and 3 ′ PCR primer complementary to the vpr terminating codon and sequences flanking the codon and also containing the MluI cloning site . Sequences in the upstream direction from the initiating codon comprise a splice acceptor. The PCR product can be hydrolyzed in the presence of Mlul and inserted into the tat rev net plasmid described above after hydrolysis in the presence of Mlul.

 Alternatively, vpr amplification can be carried out in a similar way, however, PCR primers must contain restriction sites compatible with cloning into another vector so that it is expressed under the control of the second eukaryotic promoter. A cassette originating from such a plasmid, containing a second promoter, followed by a vpr coding region, then poly (A), can be obtained by hydrolysis of restriction enzymes flanking the cassette, but not wedging within it. The DNA fragment obtained must be cloned into a single site of the tat, rev, vpr plasmid dropping out of the region necessary for expression of tat rev vpr. A plasmid with two expression units is formed in this way.

Example 52. Construction of HCV and HTLV-I plasmids
A similar approach can be used to create a plasmid expressing HTLV-1 or HCV encoded proteins, with enzymatic properties necessary for the life cycle of the virus and / or for regulatory proteins of these viruses. In the case of HTLV-1, using a plasmid skeleton and a cloning strategy similar to the above, create a plasmid encoding a regulatory regulatory protein Tax. Enzymatic protein encoding HCV genes include RNA-dependent RNA polymerase, a protein with helicase / protease functions. Information about the required sequences is published and they can be obtained at the Genebank. Structural data for HTLV-1 and HCV are provided by Cann AJ, Chen ISY Virology, 2nd Edition, edited by BN Fiddr, Raven Press, Ltd., New York, 1990, and Bradley VW Transfusion Medicine Peviews 1 (2), 93-102, 1992, respectively.

Example 53. Genetic vaccination with enzymatic genes
Genetic vaccination with genes encoding proteins with enzyme function, such as the HIV pol gene, can also be an important strategy for controlling the virus, since enzymes such as Pol are necessary for the production of live virus. Without polymerase or any of its functional components, HIV is non-pathogenic and non-infectious. Similarly, enzymatic genes of other viruses, such as HBV polymerase, appear to be attractive targets for genetic vaccination. See, for example, Radziwill et al. "Mutation analysis of the hepatitis B virus P gene product. Domain structure and RNase H activity", J. Virol. 64 (2), 613-620 (1990).

 One of the reasons for the attractiveness of viral enzymes as immunological targets is the limited ability of the amino acid sequences of such enzymes to mutate while retaining their enzymatic ability. For example, in the case of HIV-1, the Pol enzyme is characterized by a limited number of “escaped” mutations associated with resistance to nucleotide analogues, for example, AZT. However, the vast majority of immunological targets in the protein are retained even in the mutants escaped from the drug.

Example 54. Construction of HBV polymerase plasmid
The experiments reported in the literature indicate that the expression of HBV polymerase is achieved in tissue culture of cells in the presence of both a core and a polymerase open reading frame in the mRNA molecule. It was also shown that in such a situation, a mutation in the nuclear exchange of the nucleus does not affect the expression of polymerase.

 The HBV genome is amplified from a plasmid containing a head-to-tail dimer of the ADW HBV strain. Since it is desirable to express only the polymerase, but not the nucleus, the created 5 'PSR primer leads to a mutation of the translational initiating codons of the nucleus and nucleus. In addition, such a primer also introduces a mutant DRI sequence in order to exclude the possibility of the formation of replication-competent HBV genomic RNA. This PCR product is placed in a plasmid containing the kanamycin resistance gene and pBR322 origin of replication. In addition, such a plasmid contains a cytomegalovirus promoter, Payza sarcoma virus enhancer, and OB40 polyadenylation signal. To prevent the expression of HBV and X gene products, translational initiation codons for the surface antigen and the product of the X coding region are mutated.

 According to another approach to achieve expression of the HBV polymerase, PCR product encoding the coding region of the complete polymerase is amplified and cloned into a vector containing the kanamycin resistance gene and pBR322 origin of replication. In addition, such a plasmid contains a cytomegalovirus promoter, an Routh sarcoma enhancer, and an OB40 polyadenylation signal. The 5 'PCR primer for such amplification contains a cloning site and spans the translational initiation codon of the polymerase gene. The 3 'PCR product contains a restriction site for cloning the insert into the expression vector and is also complementary to the traditional termination codon of the HBV polymerase gene and the sequences flanking this termination codon. After ligation of the indicated PCR product into a plasmid containing the kanamycin resistance gene, pBR322 origin of replication, cytomegalovirus promoter, Routh sarcoma virus enhancer and OB40 polyadenylation signal, translational initiation codons for hepatitis B surface antigen and X genes are mutated to prevent the expression of these g . The alternative strategy used is similar to the above, however, the 3 'PCR primer in this case includes the HBV field (A) signal and the sequences flanking this signal. Such a 3 'primer is used if sequences comprising and / or surrounding HBV fields (A) are important for expression. Mutation analysis showed that the functioning of the HBV polymerase gene product can be excluded by replacing a particular nucleotide (Radziwell G. et al. (1990) J. Virol. 64 (2), 613-620). Before consuming a plasmid constructed in the manner described above, the expressed polymerase can be mutated by creating one of these mutations or other similar mutations.

Example 55
Granulocyte macrophage colony stimulating factor (GM-CSF) has a stimulating effect on a variety of cell lines, including neutrophils, monocytes / macrophages and eosinophils. The action of GM-CSF makes it an attractive therapeutic model. GM-CSF is approved by the FDA for use in autologous bone marrow transplantation, and clinical trials of factor effectiveness in the treatment of various neutropenia have begun. GM-CSF is currently administered as a protein, usually requiring multiple doses. It is necessary to obtain and purify such proteins.

 An alternative approach to the use of GM-CSF protein is the direct introduction of a gene construct containing a gene encoding GM-CSF, in combination with the introduction of bupivacaine. Genetically, the construct is created by PCR from a GM-CSF gene including a signal sequence. The genetic construct preferably contains a kanamycin resistance gene (aminoglycoside-3'phosphotransferase gene), a bacterial origin of replication, sequences supporting the expression of the GM-CSF coding region in cells into which the plasmid is introduced, for example, vectors characterized as skeletons in Example 46. The plasmid preferably contains the origin of mammalian replication induced by cell replication associated with the introduction of bupivacaine. In the case of the use of EBV, the origin of replication also includes the sequence encoding the nuclear antigen EBNA-1, together with the corresponding regulatory sequences. Primers for PCR amplification of the insert are restriction enzyme sites that allow cloning into the expression vector, and are complementary to the 5 ′ and 3 ′ ends of the GM-CSF coding sequence. The PCR reaction is carried out with a cDNA clone according to the method of Lee and others, Proc. Natl. Acad Sci. U.S. 82, 4360-4344.

Example 56
Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells associated with the Philadelphia chromosome, i.e. the chromosome resulting from translocation between chromosomes 9 and 22. Point discontinuity in chromosome 22 forms a cluster in a 6 kb region called the point discontinuity cluster region (COCR), while point discontinuities in chromosome 9 are scattered throughout the region in 90 K. about. upstream from c-abl exon 2. The various occurring 9:22 translocations can be divided into two types: K28 translocations and L6 translocations. Transcription via bcr-abl leads to the formation of fused mRNA. An antisense molecule targeting the bcr-abl fusion of mRNA has been shown to reduce the ability of blood-forming cells from CML patients to form colonies.

 A genetic construct encoding an antisense molecule is introduced together with bupivacaine into the cells of an individual suffering from CML ex vivo. The treated cells are then reintroduced to the individual.

Example 57
Gene constructs useful in pharmaceutical kits and preparations for HBV vaccination and treatment are constructed with vectors characterized as skeletons in Example 46. Plasmids contain HBV structural genes, in particular genes encoding the HBV surface antigen and / or HBV core antigen, and / or HBV antigen antigen.

Example 58
Gene constructs useful in pharmaceutical kits and preparations for HCV vaccination and treatment are constructed with the vectors characterized as the skeleton in Example 46. Plasmids contain HCV structural genes, in particular genes encoding the HCV core protein and / or HCV coat protein.

Example 59
The pREV gene construct is created containing the nucleotide sequence encoding HIV rev as the sole target protein. The rev coding sequence is cloned into the skeleton B described in Example 46 from BBB35 (RD System Inc., Minneapolis, MH) containing the rev coding region from the HIV strain HX3B in pUC19.

TABLE 1
The picornavirus family.

Kind
Rhinoviruses: (medicine) are responsible for ≈50% of common colds.

 Eteroviruses: (medicine) include polioviruses, coxsackie virus, echoviruses and human enteroviruses, such as hepatitis A.

 Aptoviruses: (veterinary medicine) include foot and mouth disease viruses.

 Target antigens: VP1, VP2, VP3, VP4, VPG.

 The family of calciviruses.

Kind
Norwalk Group viruses: (medicine) viruses are important causative agents of epidemic gastroenteritis.

 The togavirus family.

Kind
Alphaviruses: (medicine and veterinary medicine) examples include premature aging virus, Ross River virus, equine and western encephalitis viruses in horses.

 Reovirus: (medicine) rubella virus.

 Family of flaviruses.

 Examples include: (medicine) dengue virus, yellow fever, Japanese encephalitis, St. Louis encephalitis and tick-borne encephalitis.

 Hepatitis C virus: (medicine), these viruses have not been grouped to date, but are thought to be either togaviruses or flaviviruses. The greatest resemblance to the togavirus family.

Coronavirus Family:
(medicine and veterinary medicine) infectious bronchitis virus (poultry), pigs (pigs) gastroenteritis virus, pigs (pigs) hemagglutinating encephalomyelitis virus, feline infectious peritonitis virus (cats), feline abdominal coronavirus (cats), canine coronavirus (dogs). Human respiratory coronaviruses cause ≈40% of cases of common colds, for example, 22KE, OSK3.

 Note: Coronaviruses can cause non-A, B, or C hepatitis.

Target antigens:
E1, also called M or matrix protein; E2, also called S or spike protein; E3, also called HE or hemagglutinelteric glycoprotein (not present in all coronaviruses; N or nucleocapsid.

 The rhabdovirus family.

 Genus: vesiliovirus, lysavirus (medicine and veterinary medicine): rabies.

 Target antigen: G-protein, N-protein.

 The family of filoviruses.

Kind
Paramyxovirus: (medicine and veterinary medicine) epidemic paratitis virus, Newcastle disease virus (an important pathogen in chickens).

 Morbillivirus: (medicine and veterinary medicine) measles, canine distemper.

 Pneuminvirus: (medicine and veterinary medicine) respiratory syncytial virus.

 Orthomyxovirus family (medicine): influenza virus.

 Bunavirus Family

Kind
Bunyavirus (medicine): California encephalitis, LA Cross.

 Phlebovirus (medicine): Rift Valley Fever virus.

 Hantavirus: Puremala, a hemahagin fever virus.

 Nairvirus (veterinary medicine): Nairobi sheep disease.

 In addition, many unclassified bunyaviruses.

 The arenovirus family.

 (medicine): LCM, Lassa fever virus.

 Reovirus family.

Kind
Reovirus (a possible human pathogen).

 Rotavirus: acute gastroenteritis in children.

 Orbiviruses (medicine and veterinary medicine): Colorado tick-borne fever, lembo (human), equine encephalitis, blue tongue disease.

 The family of retroviruses.

 Subfamily: oncovirus (veterinary medicine): feline leukemia virus, HTLV-1, HTLV-2.

 Lentivirus (medicine and veterinary medicine): HIV, cat immunodeficiency virus, equine infections, anemia virus.

 Spumavirus.

 Papovavirus family.

 Subfamilies: poliomaviruses (medicine): BKU and JCU viruses.

 Papillomavirus (medicine): many viral types associated with cancer or malignant papilloma.

 Adenovirus (medicine): e.g. : AD7, ARD, A.V. - cause respiratory diseases, some adenoviruses, for example, 275 cause enteritis.

 Parvovirus family (veterinary medicine): feline parvovirus, causes enteritis in cats; feline panleukopenia virus of cats; canine parvovirus; pig parvovirus.

 The family of herpes viruses.

 Subfamily: alpha-herpes-free.

 Genus: simplex virus (medicine); HSVI, HSVII; varicellovirus (medicine and veterinary medicine): pseudorabies - chicken pox.

 Subfamily: beta-herpes-sviridny.

 Genus: cytomegalovirus; HCMV; muramegalovirus.

 Subfamily: gamma herpes sviridny.

 Genus: (medicine) lymphocryptovirus; EBV (burkitt lymph); radinovirus.

 Poxvirus family.

Subfamily: chordopoxvirid (medicine and veterinary medicine)
Genus variola (smallpox), vaccine (cowpox); parapoxvirus (veterinary medicine); auipoxvirus (veterinary medicine); capricoxvirus; leporipoxvirus, suipoxvirus.

 Subfamily: entemopoxvirid.

 Hepadnevirus family: hepatitis B virus.

 Unclassified: hepatitis delta virus.

TABLE 2
Bacterial pathogens
Pathogenic gram-positive cocci include: pneumococcus, staphylococcus, streptococcus. Pathogenic gram-negative cocci include: meningococcus and gonococcus.

 Pathogenic enteric gram-negative bacilli include: enterobacteria, pseudomonads, acinetobacteria and eikenella, melioidosis, salmonella, shigellosis, hemophilia, chancroid, brucellosis, tularemia, yersinia, montrecylocytofibliocytophyllocytobacterium, , donovanosis (inguinal granuloma) and barthenellosis.

 Pathogenic anaerobic bacteria include: tetanus, botulism, other clostridia, tuberculosis, leprosy and other microbacteria. Pathogenic spirochete diseases include: syphilis, treponematosis, tropical granuloma, pint, endemic syphilis and leptospirosis. Other infections caused by higher pathogenic bacteria and pathogenic fungi include actinomycosis, nocardiosis, cryptococcosis, blastomycosis, histoplasmosis, mucormycosis, sporotrichosis, paracoccidiomycosis, petriellidiosis, torulopsosis, mycetoma, and chromomycosis.

 Rickettsial infections include rickettsiosis.

 Examples of mycoplasmic and chlamydial infections include: mycoplasmic pneumonia, venereal lymphogranuloma, psitacosis, and perinatal chlamydial infections.

Pathogenic eukaryotes
Pathogenic protozoa and helminths and the infections they create include: amoebiasis, malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, pneumocystis cariniosis, babysiosis, giardiasis, trichinosis, filariosis, schistosomiasis, nematodes, trematodes or trematodes (trematodes and trematodes).

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TTGTTTAACT TTTGATCGAT ССАТТСС
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GATTTGTATC GATGATCTGA С
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TGTAGTAGCA AAAGAAATAG TTAAG
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ААТТСТТААС ТАТТТСТТТТ GCTAC
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ATTTGTCGAC TGGTTTCAGC CTGCCATGGC AGGAAGAAGC
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ACGACGCGTA TTCTTTAGCT CCTGACTCC
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GCTGACGGTA GCGGCCGCAC ААТТ
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GTATTAAGCG GCCGCAATTG ТТ
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AAAAAGCTTC GCGGATCCGC GTTGCGGCCG CAACCGGTCA CCGGCGACGC GTCGGTCGAC CGGTCATGGC TGGGCCCC
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CCCAGCTTA GACATGATAA GATACATTG
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CTAGCAGCTG GATCCCAGCT TC
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GGATTTCTGG GGATCCAAGC TAGT
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TATAGGATCC GCGCAATGAA AGACCCCACC Т
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ATATGGATCC GCAATGAAAG ACCCCCGCTG А
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TAAAGCGGCC GCTCCTATGG CAGGAAGACG
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ATTACGCGTC TTATGCTTCT AGCCAGGCAC AATG
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ATTACGCGTT TATTACAGAA TGGAAAACAG ATGGCAGGTG
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ATTACGCGTT ATTGCAGAAT TCTTATTATG GC
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GAGGCTTGGA GAGGATTATA GAAGTACTGC AAGAGCTG
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GAATCCTCTC CAAGCCTCAG CTACTGCTAT AGCTGTGGC
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(XI) Описание последовательности: ПОСЛЕД. N 35:
AAAAATAAAG CGGCCGCTCC TATGGCAGGA AGAGAAGCG
(2) Информация для ПОСЛЕД. N 36:
(I) Характеристики последовательности:
(А) Длина: 39 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 36:
ААААААТТАС GCGTCTTATG CTTCTAGCCA GGCACAATG
(2) Информация для ПОСЛЕД. N 37:
(I) Характеристики последовательности:
(А) Длина: 31 пара оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 37:
CCCAAGCTTG GGAATGCTCT GCCAGTGTTA С
(2) Информация для ПОСЛЕД. N 38:
(I) Характеристики последовательности:
(А) Длина: 36 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 38:
GGGGGCCGGA AGGGCACAAT AAAACTGTCT GCTTAC
(2) Информация для ПОСЛЕД. N 39:
(I) Характеристики последовательности:
(А) Длина: 33 пары оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 39:
CCTGATTCAG GTGAAAATAT TGTTGATGCG CTG
(2) Информация для ПОСЛЕД. N 40:
(I) Характеристики последовательности:
(А) Длина: 111 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 40:
ААСАТСААТА СААССТАТТА АТТТССССТС GTCAAAAATA AGGTTATCAA GTGAGAAATC ACCATCAGTG ACGACTGAAT CCGGTGAGAA TGGCAAAAGT TTATGCATTT С
(2) Информация для ПОСЛЕД. N 41:
(I) Характеристики последовательности:
(А) Длина: 55 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 41:
CTAGCTCGGG GATCCGCGTT GCGGCCGCAA AAAGTCGACG GGCGACGCGT AAAAA
(2) Информация для ПОСЛЕД. N 42:
(I) Характеристики последовательности:
(А) Длина: 55 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 42:
GATCTTTTTA CGCGTCGCCC GTCGACTTTT TGCGGCCGCA ACGCGGATCC CCGCG
(2) Информация для ПОСЛЕД. N 43:
(I) Характеристики последовательности:
(А) Длина: 48 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 43:
ATGTCGACTG GTTTCAGCCT GCCATGGCAG GAAGAAGC
(2) Информация для ПОСЛЕД. N 44:
(I) Характеристики последовательности:
(А) Длина: 35 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 44:
CCCCACGACG CGTCTATTCT TTAGCTCCTG АСТСС
(2) Информация для ПОСЛЕД. N 45:
(I) Характеристики последовательности:
(А) Длина: 35 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 45:
TTTGCGGCCG CGTAAGTGGA GAGAGATGGT GCGAG
(2) Информация для ПОСЛЕД. N 46:
(1) Характеристики последовательности
(А) Длина: 21 пара оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 46:
CTGGTGGGGC TGTTGGCTCT G
(2) Информация для ПОСЛЕД. N 47:
(I) Характеристики последовательности:
(А) Длина: 80 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 47:
ААТТТААТАА GTAAGTAAGT GTCATATGTT TGTTTGAATT CTGCAACAAC TGCTGTTTAT CCATTTTCAG AATTGGGTG
(2) Информация для ПОСЛЕД. N 48:
(I) Характеристики последовательности:
(А) Длина: 80 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 48:
TCGACACCCA ATTCTGAAAA TGGATAAACA GCACTTGTTG CAGAATTCAA АСАААСАТАТ GACACTTACT ТАСТТАТТА
(2) Информация для ПОСЛЕД. N 49:
(I) Характеристики последовательности:
(А) Длина: 41 пара оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 49:
GGGGTTTTTG GGCATATGTA TGAGGGACAA TTGGAGAAGT G
(2) Информация для ПОСЛЕД. N 50
(I) Характеристики последовательности:
(А) Длина: 70 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 50:
AAGCTTGTGG AATTCTTAAT TTCTCTGTCC GGGGTTTTTG GGCATATGTA TGAGGGACAT TGGAGAAGTG
(2) Информация для ПОСЛЕД. N 51:
(I) Характеристики последовательности:
(А) Длина: 29 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 51:
CAGTATCTGG CATGGGTAC
(2) Информация для ПОСЛЕД. N 52:
(I) Характеристики последовательности:
(А) Длина: 29 пар оснований
(В) Тип: нуклеиновая кислота
(С) Число нитей: одна
(D) Топология: линейная
(II) Тип молекулы: ДНК
(XI) Описание последовательности: ПОСЛЕД. N 52:
CCATGCCAGA TACTGGTACйLIST OF SEQUENCES
(2) Information for the AFTER. N 1:
(I) Characteristics of the sequence
(A) Length :; 32 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(Ix) Description of sequence: LAST. N 1:
AGGCGTCTCG AGACAGAGGA GAGCAAGAAA TG
(2) Information for the AFTER. N 2:
(I) Characteristics of the sequence
(A) Length: 30 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 2:
TTTSSSTSTA GATAAGCCAT CCAATSASAS
(2) Information for the AFTER. N 3:
(I) Characteristics of the sequence:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 3:
GAAGGATCCA TGAAAAAATA TTTATTGGG
(2) Description for LAST. N 4:
(I) Characteristics of the sequence:
(A) Length: 30 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 4:
ACTGTCGACT TATTTTAAAG CGTTTTTAAG
(2) Information for the AFTER. N 5:
(I) Characteristics of the sequence:
(A) Length: 30 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 5:
GCCAGTTTTG GATCCTTAAA AAAGGCTTGG
(2) Information for the AFTER. N 6:
(I) Characteristics of the sequence:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 6:
TTGTGAGGGA CAGAATTCCA ATCAGGG
(2) Information for the AFTER. N 7:
(I) Characteristics of the sequence:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 7:
CAGTGATATC CCGGGAGACT CCTC
(2) Information for the AFTER. N 8:
(I) Characteristics of the sequence:
(A) Length: 25 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 8:
GAATAGAAGA ACTCCTCTAG AATTS
(2) Information for the AFTER. N 9:
(I) Characteristics of the sequence:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 9:
GCCTTAGGCG GATCCTATGG CAGGAAG
(2) Information for the AFTER. N 10:
(I) Characteristics of the sequence:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 10:
TAAGATGGGT GGCCATGGTG AATT
(2) Information for the AFTER. N 11:
(I) Characteristics of the sequence:
(A) Length: 22 amino acids
(B) Type: amino acid
(D) Topology: linear
(II) Molecule Type: Peptide
(XI) Description of the sequence: LAST. N 11:

(2) Information for the AFTER. N 12:
(I) Characteristics of the sequence:
(A) Length: 25 amino acids
(B) Type: amino acid
(D) Topology: linear
(II) Molecule Type: Peptide
(XI) Description of the sequence: LAST. N 12:

(2) Information for the AFTER. N 13:
(I) Characteristics of the sequence:
(A) Length: 27 amino acids
(B) Type: amino acid
(D) Topology: linear
(II) Molecule Type: Peptide
(XI) Description of the sequence: LAST. N 13:

(2) Information for the AFTER. N 14:
(I) Characteristics of the sequence:
(A) Length: 25 amino acids
(B) Type: amino acid
(D) Topology: linear
(II) Molecule Type: Peptide
(XI) Description of the sequence: LAST. N 14:

(2) Information for the AFTER. N 15:
(I) Characteristics of the sequence:
(A) Length: 27 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 15:
TTGTTTAACT TTTGATCGAT SSATTSS
(2) Information for the AFTER. N 16:
(I) Characteristics of the sequence:
(A) Length: 21 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 16:
GATTTGTATC GATGATCTGA C
(2) Information for the AFTER. N 17:
(I) Characteristics of the sequence:
(A) Length: 25 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 17:
TGTAGTAGCA AAAGAAATAG TTAAG
(2) Information for the AFTER. N 18:
(I) Characteristics of the sequence:
(A) Length: 25 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 18:
AATTSTTAAS TATTTSTTTT GCTAC
(2) Information for the AFTER. N 19:
(I) Characteristics of the sequence:
(A) Length: 40 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 19:
ATTTGTCGAC TGGTTTCAGC CTGCCATGGC AGGAAGAAGC
(2) Information for the AFTER. N 20:
(I) Characteristics of the sequence:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 20:
ACGACGCGTA TTCTTTAGCT CCTGACTCC
(2) Information for the AFTER. N 21:
(I) Characteristics of the sequence:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 21:
GCTGACGGTA GCGGCCGCAC AATT
(2) Information for the AFTER. N 22:
(I) Characteristics of the sequence:
(A) Length: 22 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 22:
GTATTAAGCG GCCGCAATTG TT
(2) Information for the AFTER. N 23:
(I) Characteristics of the sequence:
(A) Length: 78 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 23:
AAAAAGCTTC GCGGATCCGC GTTGCGGCCG CAACCGGTCA CCGGCGACGC GTCGGTCGAC CGGTCATGGC TGGGCCCC
(2) Information for the AFTER. N 24:
(I) Characteristics of the sequence:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 24:
CCCAGCTTA GACATGATAA GATACATTG
(2) Information for the AFTER. N 25:
(I) Characteristics of the sequence:
(A) Length: 22 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 25:
CTAGCAGCTG GATCCCAGCT TC
(2) Information for the AFTER. N 26:
(I) Characteristics of the sequence:
(A) Length: 24 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 26:
GGATTTCTGG GGATCCAAGC TAGT
(2) Information for the AFTER. N 27:
(I) Characteristics of the sequence:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 27:
TATAGGATCC GCGCAATGAA AGACCCCACC T
(2) Information for the AFTER. N 28:
(I) Characteristics of the sequence:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 28:
ATATGGATCC GCAATGAAAG ACCCCCGCTG A
(2) Information for the AFTER. N 29:
(I) Characteristics of the sequence:
(A) Length: 30 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 29:
TAAAGCGGCC GCTCCTATGG CAGGAAGACG
(2) Information for the AFTER. N 30:
(I) Characteristics of the sequence:
(A) Length: 34 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 30:
ATTACGCGTC TTATGCTTCT AGCCAGGCAC AATG
(2) Information for the AFTER. N 31:
(I) Characteristics of the sequence:
(A) Length: 40 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 31:
ATTACGCGTT TATTACAGAA TGGAAAACAG ATGGCAGGTG
(2) Information for the AFTER. N 32:
(I) Characteristics of the sequence:
(A) Length; 32 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 32:
ATTACGCGTT ATTGCAGAAT TCTTATTATG GC
(2) Information for the AFTER. N 33:
(I) Characteristics of the sequence:
(A) Length: 38 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 33:
GAGGCTTGGA GAGGATTATA GAAGTACTGC AAGAGCTG
(2) Information for the AFTER. N 34:
(I) Characteristics of the sequence:
(A) Length: 39 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 34:
GAATCCTCTC CAAGCCTCAG CTACTGCTAT AGCTGTGGC
(2) Information for the AFTER. N 35:
(I) Characteristics of the sequence:
(A) Length: 39 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 35:
AAAAATAAAG CGGCCGCTCC TATGGCAGGA AGAGAAGCG
(2) Information for the AFTER. N 36:
(I) Characteristics of the sequence:
(A) Length: 39 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 36:
AAAAAATTAS GCGTCTTATG CTTCTAGCCA GGCACAATG
(2) Information for the AFTER. N 37:
(I) Characteristics of the sequence:
(A) Length: 31 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 37:
CCCAAGCTTG GGAATGCTCT GCCAGTGTTA C
(2) Information for the AFTER. N 38:
(I) Characteristics of the sequence:
(A) Length: 36 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 38:
GGGGGCCGGA AGGGCACAAT AAAACTGTCT GCTTAC
(2) Information for the AFTER. N 39:
(I) Characteristics of the sequence:
(A) Length: 33 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 39:
CCTGATTCAG GTGAAAATAT TGTTGATGCG CTG
(2) Information for the AFTER. N 40:
(I) Characteristics of the sequence:
(A) Length: 111 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 40:
AASATSAATA SAASSTATTA ATTTSSSSTS GTCAAAAATA AGGTTATCAA GTGAGAAATC ACCATCAGTG ACGACTGAAT CCGGTGAGAA TGGCAAAAGT TTATGCATTT C
(2) Information for the AFTER. N 41:
(I) Characteristics of the sequence:
(A) Length: 55 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 41:
CTAGCTCGGG GATCCGCGTT GCGGCCGCAA AAAGTCGACG GGCGACGCGT AAAAA
(2) Information for the AFTER. N 42:
(I) Characteristics of the sequence:
(A) Length: 55 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 42:
GATCTTTTTA CGCGTCGCCC GTCGACTTTT TGCGGCCGCA ACGCGGATCC CCGCG
(2) Information for the AFTER. N 43:
(I) Characteristics of the sequence:
(A) Length: 48 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 43:
ATGTCGACTG GTTTCAGCCT GCCATGGCAG GAAGAAGC
(2) Information for the AFTER. N 44:
(I) Characteristics of the sequence:
(A) Length: 35 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 44:
AC CCCC AC AC AC AC AC AC AC AC CCCC CCCC CCCC CCCC CCCC CCCC CCCC CCCC CCCC AC
(2) Information for the AFTER. N 45:
(I) Characteristics of the sequence:
(A) Length: 35 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 45:
TTTGCGGCCG CGTAAGTGGA GAGAGATGGT GCGAG
(2) Information for the AFTER. N 46:
(1) Characteristics of the sequence
(A) Length: 21 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 46:
CTGGTGGGGC TGTTGGCTCT G
(2) Information for the AFTER. N 47:
(I) Characteristics of the sequence:
(A) Length: 80 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 47:
AATTTAATAA GTAAGTAAGT GTCATATGTT TGTTTGAATT CTGCAACAAC TGCTGTTTAT CCATTTTCAG AATTGGGTG
(2) Information for the AFTER. N 48:
(I) Characteristics of the sequence:
(A) Length: 80 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 48:
TCGACACCCA ATTCTGAAAA TGGATAAACA GCACTTGTTG CAGAATTCAA ASAAASATAT GACACTTACT TASTATTA
(2) Information for the AFTER. N 49:
(I) Characteristics of the sequence:
(A) Length: 41 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 49:
GGGGTTTTTTG GGCATATGTA TGAGGGACAA TTGGAGAAGT G
(2) Information for the AFTER. N 50
(I) Characteristics of the sequence:
(A) Length: 70 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 50:
AAGCTTGTGG AATTCTTAAT TTCTCTGTCC GGGGTTTTTG GGCATATGTA TGAGGGACAT TGGAGAAGTG
(2) Information for the AFTER. N 51:
(I) Characteristics of the sequence:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 51:
CAGTATCTGG CATGGGTAC
(2) Information for the AFTER. N 52:
(I) Characteristics of the sequence:
(A) Length: 29 base pairs
(B) Type: nucleic acid
(C) Number of threads: one
(D) Topology: linear
(II) Molecule Type: DNA
(XI) Description of the sequence: LAST. N 52:
CCATGCCAGA TACTGGTAC

Claims (48)

 1. The method of introducing into an individual's cells genetic material, characterized in that the cells are contacted with an enhancer of polynucleotide function and nucleic acid molecules containing no retrovirus particles are introduced into the cells.  2. The method according to claim 1, characterized in that the polynucleotide function enhancer is represented by bupivacaine.  3. The method according to p. 1, characterized in that the nucleic acid molecule comprises a nucleotide sequence encoding a protein and operably linked to regulatory sequences.  4. The method according to p. 1, characterized in that the nucleic acid molecule comprises a nucleotide sequence encoding a protein, which includes at least one epitope that is identical or substantially similar to the epitope of the antigen against which an immune response is desired, the nucleotide the sequence is operably linked to regulatory sequences.  5. The method according to p. 1, characterized in that the nucleic acid molecule includes a nucleotide sequence encoding a protein, which includes at least one epitope that is identical or essentially similar to the epitope of the pathogen antigen.  6. The method according to claim 5, characterized in that the polynucleotide function enhancer is represented by bupivacaine.  7. The method according to p. 5, characterized in that the nucleic acid molecule is a DNA molecule.  8. The method according to claim 5, characterized in that the protein is represented by a pathogen antigen or fragment thereof.  9. The method according to p. 5, characterized in that the nucleic acid molecule is administered intramuscularly.  10. The method according to claim 5, characterized in that the pathogen is a virus selected from the group comprising the human immunodeficiency virus (HIV); human T-cell leukemia virus (TRVL), influenza virus, hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), herpes simplex virus 1 (HSV1), virus herpes simplex 2 (HSV2), cytomegalovirus (CMV), Epstein-Barr virus (NBV), rhinovirus and coronavirus.  11. The method according to claim 5, characterized in that the pathogen is HIV and the nucleic acid molecule comprises a nucleotide sequence encoding an HIV protein.  12. The method according to claim 5, characterized in that the pathogen is represented by HIV and the nucleic acid molecule comprises nucleotide sequences encoding more than one structural HIV protein.  13. The method according to claim 5, characterized in that the pathogen is HIV and the nucleic acid molecule comprises nucleotide sequences encoding more than one regular HIV protein.  14. The method according to claim 5, characterized in that at least two or more different nucleic acid molecules are introduced into different cells of the individual, and different nucleic acids include each nucleotide sequence encoding one or more antigens of the same pathogen.  15. The method according to claim 5, characterized in that the polynucleotide function enhancer and the nucleic acid molecule are administered simultaneously.  16. The method according to claim 5, characterized in that the individual is a person; the polynucdeotide function enhancer is represented by bupivacaine; the pathogen is a human immunodeficiency virus; the nucleic acid molecule is represented by DNA and includes a DNA sequence encoding the gag and pol structural proteins of HIV with a psi deletion, the gag and pol encoding DNA sequence operably linked to an Routh sarcoma virus enhancer, directly an early cytomegalovirus promoter, and a small SV 40 polyadenylation signal and optionally SV 40 replication start point.  17. The method according to clause 16, wherein the DNA sequence further includes a rev-responsive element of HIV and a deletion of HIV integrase.  18. The method according to 17, characterized in that the DNA sequence further includes a splice acceptor of HIV.  19. The method of claim 16, wherein the DNA molecule further comprises a DNA sequence encoding a sequence additionally containing HIV rev, operably linked to the SV 40 promoter and the SV 40 small polyadenylation signal and optionally to the SV 40 origin of replication.  20. The method according to claim 19, wherein the rev coding DNA sequence encodes furthermore HIV vpu and HIV env.  21. The method according to claim 19, wherein the DNA sequence encoding gag and pol further comprises a rev-responsive HIV element and a deletion of HIV integrase.  22. The method according to item 21, wherein the DNA sequence encoding gag and pol, further includes a splice-promoter of HIV.  23. The method according to claim 5, characterized in that the individual is a human polynucleotide function enhancer represented by bupivacaine; the pathogen is a human immunodeficiency virus; the nucleic acid molecule is DNA and contains a DNA sequence encoding the H1V rev, vpu, and env proteins operably linked to the Routh sarcoma virus enhancer, the immediate early cytomegalovirus promoter, and the small SV 40 polyadenylation signal and optionally the SV 40 origin of replication.  24. The method according to claim 5, characterized in that the individual is a person, the polynucleotide function enhancer is represented by bupivacaine; the pathogen is a human immunodeficiency virus, two different DNA molecules are introduced into different cells of an individual, the molecule of one of the nucleic acids being DNA and includes a DNA sequence encoding the gag and pol structural proteins of HIV with a psi deletion, and the DNA sequence encoding gag and pol , operably linked to an Routh sarcoma virus enhancer, directly an early cytomegalovirus promoter and a small SV 40 polyadenylation signal, and optionally with an SV 40 replication origin, another nucleic acid molecule This is DNA and includes the DNA sequence encoding the HIV proteins rev, vpu and env operably linked to the Routh sarcoma virus enhancer, directly the early cytomegalovirus promoter and the small polyadenylation signal SV 40 and optionally with the SV 40 origin of replication.  25. The method according to claim 5, characterized in that the implementation of the introduction into human cells of molecules of two different nucleic acids, the molecule of each nucleic acid comprising a nucleotide sequence encoding a protein containing at least one epitope that is identical or essentially similar an epitope of at least one HIV antigen operably linked to regulatory sequences, the nucleotide sequences being able to be expressed in cells, the nucleotide sequences of each of two times s nucleic acids encode different proteins, these proteins have at least an epitope identical or substantially similar to an epitope of at least one HIV protein encoded by HIV genes selected from the group consisting of gag, pol and env.  26. The method according to claim 1, characterized in that the nucleic acid molecule comprises a nucleotide sequence encoding a target protein containing an epitope that is identical or essentially similar to the epitope of a protein associated with cells characterizing the disease.  27. The method according to p, characterized in that the polynucleotide function enhancer is represented by bupivacaine.  28. The method according to p, characterized in that the disease is characterized by hyperproliferation of cells.  29. The method according to p, characterized in that the disease is an autoimmune disease.  30. The method according to p, characterized in that the nucleic acid molecule is a DNA molecule.  31. The method according to p, characterized in that the nucleic acid molecule is administered intramuscularly.  32. The method according to p. 26, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a target protein selected from the group consisting of protein products of the oncogenes myb, myc, fyn, ras, sarc, neu and trk; protein products of the bcl / abl translocation gene; P53; EGRF; the variable regions of antibodies obtained using B-cell lymphomas; and the variable regions of T-cell receptors of T-cell lymphomas.  33. The method according to p. 26, wherein the protein is selected from the group comprising variable regions of antibodies involved in B-cell-mediated autoimmune disease and variable regions of T-cell receptors involved in T-cell-mediated autoimmune disease.  34. The method according to claim 1, characterized in that the nucleic acid molecule comprises a nucleotide sequence encoding a protein, the presence of which will compensate for an absent, non-functional or partially functional protein or provide a therapeutic effect in an individual.  35. The method according to clause 34, wherein the polynucleotide function enhancer is represented by bupivacaine.  36. The method according to clause 34, wherein the nucleic acid molecule is a DNA molecule.  37. The method according to clause 34, wherein the nucleic acid molecule is administered intramuscularly.  38. The method according to clause 34, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a protein selected from the group consisting of enzymes, structural proteins, cytokines, lymphokines and growth factors.  39. A pharmaceutical composition for introducing genetic material that encodes HIV proteins into an individual's cage, comprising a nucleic acid molecule represented by a DNA molecule that includes a DNA sequence encoding a protein, optionally linked to regulatory elements, and a polynucleotide function enhancer.  40. The pharmaceutical composition according to § 39, wherein the nucleic acid molecule comprises a nucleotide sequence encoding a protein selected from the group consisting of proteins comprising at least one epitope identical or substantially similar to an epitope of a pathogen antigen; proteins containing an epitope identical to or essentially similar to an epitope of a protein associated with hyperproliferating cells; proteins containing an epitope that is identical or substantially similar to an epitope of a protein associated with cells characterizing an autoimmune disease; proteins whose presence will compensate for an absent non-functional or partially functioning protein in an individual; and proteins that provide a therapeutic effect in an individual.  41. The pharmaceutical composition according to § 39, wherein the pathogen is a virus selected from the group consisting of human immunodeficiency virus (HIV); human T-cell leukemia virus (HTLV); influenza virus; hepatitis A virus (HAV); hepatitis B virus (HBV); hepatitis C virus (HCV); human papillomavirus (HPV); herpes simplex virus 1 (HSV1); herpes simplex virus 2 (HSV2); cytomegalovirus (CMV); Epstein-Barr virus (EBV); rhinovirus and coronavirus.  42. The pharmaceutical composition according to § 39, wherein the nucleic acid molecule is a DNA sequence encoding the HIV gag and pol structural proteins with a psi deletion, the DNA sequence encoding gag and pol operably linked to an Routh sarcoma virus enhancer, directly by the early cytomegalovirus promoter, small SV 40 polyadenylation signal, and optionally with SV.40 replication origin.  43. The pharmaceutical composition according to claim 39, wherein the nucleic acid molecule is a DNA molecule comprising a DNA sequence encoding HIV rev, vpu and env proteins operably linked to an Routh sarcoma virus enhancer, directly an early cytomegalovirus promoter, a small polyadenylation signal SV 40 and optionally SV 40 replication origin.  44. The pharmaceutical composition according to § 39, wherein the polynucleotide function enhancer is represented by bupivacaine.  45. A pharmaceutical kit for introducing genetic material that encodes HIV proteins into cells of an individual, comprising a first inoculum containing a pharmaceutically acceptable carrier or diluent and a first nucleic acid molecule comprising a nucleotide sequence encoding at least one HIV protein operably linked to regulatory sequences, the nucleic sequence being able to be expressed in human cells, and a second inoculum containing a pharmaceutically acceptable n a carrier or diluent and a second nucleic acid molecule comprising a nucleotide sequence encoding at least one HIV protein operably linked to regulatory sequences, the nucleotide sequence being able to be expressed in human cells, wherein the first nucleic acid molecule is not identical to the second nucleic acid molecule.  46. The pharmaceutical kit according to item 45, wherein the first and second inoculum include a polynucleotide function enhancer or a third inoculum containing a polynucleotide function enhancer.  47. The pharmaceutical kit according to item 45, wherein the first nucleic acid molecule and the second nucleic acid molecule together encode HIV gag, pol and env proteins.  48. The pharmaceutical kit according to item 45, characterized in that it includes a first inoculum containing a first pharmaceutical composition comprising a DNA molecule that contains a DNA sequence encoding the HIV gag and pol structural proteins with psi deletion, said sequence encoding gag and pol is operably linked to the potency of the Routh sarcoma virus, directly by the early cytomegalovirus promoter, the small SV 40 polyadenylation signal, and optionally the SV 40 origin of replication, and a second inoculum containing a second pharmaceutical a chemical composition comprising a DNA molecule that contains a DNA sequence encoding HIV rev, vpu, and env proteins operably linked to an Routh sarcoma virus enhancer, directly an early cytomegalovirus promoter, a small SV 40 polyadenylation signal, and optionally an SV 40 origin of replication. Priorities for items:
01/26/1993 according to claims 2, 17, 44, 45 and 48;
03/11/1993 according to claims 6, 18 - 25 and 27.

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